Anti-VEGF antibodies

ABSTRACT

Humanized and variant anti-VEGF antibodies and various uses therefor are disclosed. The anti-VEGF antibodies have strong binding affinities for VEGF; inhibit VEGF-induced proliferation of endothelial cells in vitro; and inhibit tumor growth in vivo.

CROSS REFERENCES

This application is a continuation of U.S. application Ser. No.09/056,160 filed Apr. 6, 1998, now abandoned, which claims benefit under35 U.S.C. §119(e) of Provisional Application Ser. No. 60/126,446, filedApr. 7, 1997, and Ser. No. 60/054,856 filed Aug. 6, 1997. The disclosureof both aforementioned Applications are expressly incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to anti-VEGF antibodies and, inparticular, to humanized anti-VEGF antibodies and variant anti-VEGFantibodies.

2. Description of Related Art

It is now well established that angiogenesis is implicated in thepathogenesis of a variety of disorders. These include solid tumors,intraocular neovascular syndromes such as proliferative retinopathies orage-related macular degeneration (AMD), rheumatoid arthritis, andpsoriasis (Folkman et al. J. Biol Chem. 267:10931–10934 (1992);Klagsbrun et al Annu. Rev. Physiol. 53:217–239 (1991); and Garner A,Vascular diseases. In: Pathobiology of ocular disease. A dynamicapproach. Garner A, Klintworth G K, Eds. 2nd Edition Marcel Dekker, NY,pp 1625–1710 (1994)). In the case of solid tumors, theneovascularization allows the tumor cells to acquire a growth advantageand proliferative autonomy compared to the normal cells. Accordingly, acorrelation has been observed between density of microvessels in tumorsections and patient survival in breast cancer as well as in severalother tumors (Weidner et al. N Engl J Med 324:1–6 (1991); Horak et al.Lancet 340:1120–1124 (1992); and Macchiarini et al. Lancet 340:145–146(1992)).

The search for positive regulators of angiogenesis has yielded manycandidates, including aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-α, angiogenin,IL-8, etc. (Folkman et al. and Klagsbrun et al). The negative regulatorsso far identified include thrombospondin (Good et al. Proc. Natl. Acad.Sci. USA. 87:6624–6628 (1990)), the 16-kilodalton N-terminal fragment ofprolactin (Clapp et al. Endocrinology, 133:1292–1299 (1993)),angiostatin (O'Reilly et al. Cell, 79:315–328 (1994)) and endostatin(O'Reilly et al. Cell, 88:277–285 (1996)).

Work done over the last several years has established the key role ofvascular endothelial growth factor (VEGF) in the regulation of normaland abnormal angiogenesis (Ferrara et al. Endocr. Rev. 18:4–25 (1997)).The finding that the loss of even a single VEGF allele results inembryonic lethality points to an irreplaceable role played by thisfactor in the development and differentiation of the vascular system(Ferrara et al.). Furthermore, VEGF has been shown to be a key mediatorof neovascularization associated with tumors and intraocular disorders(Ferrara et al.). The VEGF mRNA is overexpressed by the majority ofhuman tumors examined (Berkman et al. J Clin Invest 91:153–159 (1993);Brown et al. Human Pathol. 26:86–91 (1995); Brown et al. Cancer Res.53:4727–4735 (1993); Mattern et al. Brit. J. Cancer. 73:931–934 (1996);and Dvorak et al. Am J. Pathol. 146:1029–1039 (1995)). Also, theconcentration of VEGF in eye fluids are highly correlated to thepresence of active proliferation of blood vessels in patients withdiabetic and other ischemia-related retinopathies (Aiello et al. N.Engl. J. Med. 331:1480–1487 (1994)). Furthermore, recent studies havedemonstrated the localization of VEGF in choroidal neovascular membranesin patients affected by AMD (Lopez et al. Invest. Ophtalmo. Vis. Sci.37:855–868 (1996)). Anti-VEGF neutralizing antibodies suppress thegrowth of a variety of human tumor cell lines in nude mice (Kim et al.Nature 362:841–844 (1993); Warren et al. J. Clin. Invest. 95:1789–1797(1995); Borgström et al. Cancer Res. 56:4032–4039 (1996); and Melnyk etal. Cancer Res. 56:921–924 (1996)) and also inhibit intraocularangiogenesis in models of ischemic retinal disorders (Adamis et al.Arch. Ophthalmol. 114:66–71 (1996)). Therefore, anti-VEGF monoclonalantibodies or other inhibitors of VEGF action are promising candidatesfor the treatment of solid tumors and various intraocular neovasculardisorders.

SUMMARY OF THE INVENTION

This application describes humanized anti-VEGF antibodies and anti-VEGFantibody variants with desirable properties from a therapeuticperspective, including strong binding affinity for VEGF; the ability toinhibit VEGF-induced proliferation of endothelial cells in vitro; andthe ability to inhibit VEGF-induced angiogenesis in vivo.

The preferred humanized anti-VEGF antibody or variant anti-VEGF antibodyherein binds human VEGF with a K_(d) value of no more than about 1×10⁻⁸Mand preferably no more than about 5×10⁻⁹M. In addition, the humanized orvariant anti-VEGF antibody may have an ED50 value of no more than about5 nM for inhibiting VEGF-induced proliferation of endothelial cells invitro. The humanized or variant anti-VEGF antibodies of particularinterest herein are those which inhibit at least about 50% of tumorgrowth in an A673 in vivo tumor model, at an antibody dose of 5 mg/kg.

In one embodiment, the anti-VEGF antibody has a heavy and light chainvariable domain, wherein the heavy chain variable domain compriseshypervariable regions with the following amino acid sequences: CDRH1(GYX₁FTX₂YGMN, wherein X₁ is T or D and X₂ is N or H; SEQ ID NO:130),CDRH2 (WINTYTGEPTYAADFKR; SEQ ID NO:2) and CDRH3 (YPX₁YYGX₂SHWYFDV,wherein X₁ is Y or H and X₂ is S or T; SEQ ID NO:131). For example, theheavy chain variable domain may comprise the amino acid sequences ofCDRH1 (GYTFTNYGMN; SEQ ID NO:1), CDRH2 (WINTYTGEPTYAADFKR; SEQ ID NO:2)and CDRH3 (YPHYYGSSHWYFDV; SEQ ID NO:3). Preferably, the three heavychain hypervariable regions are provided in a human framework region,e.g., as a contiguous sequence represented by the following formula:FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4.

The invention further provides an anti-VEGF antibody heavy chainvariable domain comprising the amino acid sequence:

-   EVQLVESGGGLVQPGGSLRLSCAASGYX₁FTX₂YGMNWVRQAPGKGLEWVGWINTYTGEPT    YAADFKRRFTFSLDTSKSTAYLQMNSRAEDTAVYYCAKYPX₃YYGX₄SHWYFDVWGQGTLV TVSS    (SEQ ID NO:127), wherein X₁ is T or D; X₂ is N or H; X₃ is Y or H    and X₄ is S or T. One particularly useful heavy chain variable    domain sequence is that of the F(ab)-12 humanized antibody of    Example 1 and comprises the heavy chain variable domain sequence of    SEQ ID NO:7. Such preferred heavy chain variable domain sequences    may be combined with the following preferred light chain variable    domain sequences or with other light chain variable domain    sequences, provided that the antibody so produced binds human VEGF.

The invention also provides preferred light chain variable domainsequences which may be combined with the above-identified heavy chainvariable domain sequences or with other heavy chain variable domainsequences, provided that the antibody so produced retains the ability tobind to human VEGF. For example, the light chain variable domain maycomprise hypervariable regions with the following amino acid sequences:CDRL1 (SASQDISNYLN; SEQ ID NO:4), CDRL2 (FTSSLHS; SEQ ID NO:5) and CDRL3(QQYSTVPWT; SEQ ID NO:6). Preferably, the three light chainhypervariable regions are provided in a human framework region, e.g., asa contiguous sequence represented by the following formula:FR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4.

In one embodiment, the invention provides a humanized anti-VEGF antibodylight chain variable domain comprising the amino acid sequence:

-   DIQX₁TQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFS    GSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKR (SEQ ID NO:126),    wherein X₁ is M or L. One particularly useful light chain variable    domain sequence is that of the F(ab)-12 humanized antibody of    Example 1 and comprises the light chain variable domain sequence of    SEQ ID NO:8.

The invention also provides a variant of a parent anti-VEGF antibody(which parent antibody is preferably a humanized or human anti-VEGFantibody), wherein the variant binds human VEGF and comprises an aminoacid substitution in a hypervariable region of the heavy or light chainvariable domain of the parent anti-VEGF antibody. The variant preferablyhas one or more substitution(s) in one or more hypervariable region(s)of the anti-VEGF antibody. Preferably, the substitution(s) are in theheavy chain variable domain of the parent antibody. For example, theamino acid subsition(s) may be in the CDRH1 and/or CDRH3 of the heavychain variable domain. Preferably, there are substitutions in both thesehypervariable regions. Such “affinity matured” variants are demonstratedherein to bind human VEGF more strongly than the parent anti-VEGFantibody from which they are generated, i.e., they have a K_(d) valuewhich is significantly less than that of the parent anti-VEGF antibody.Preferably, the variant has an ED50 value for inhibiting VEGF-inducedproliferation of endothelial cells in vitro which is at least about 10fold lower, preferably at least about 20 fold lower, and most preferablyat least about 50 fold lower, than that of the parent anti-VEGFantibody. One particularly prefered variant is the Y0317 variant ofExample 3, which has a CDRH1 comprising the amino acidsequence:GYDFTHYGMN (SEQ ID NO:128) and a CDRH3 comprising the aminoacid sequence:YPYYYGTSHWYFDV (SEQ ID NO:129). These hypervariableregions and CDRH2 are generally provided in a human framework region,e.g., resulting in a heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO:118. Such heavy chain variable domainsequences are optionally combined with a light chain variable domaincomprising the amino acid sequence of SEQ ID NO:126, and preferably thelight chain variable domain amino acid sequence of SEQ ID NO:117.

Various forms of the antibody are contemplated herein. For example, theanti-VEGF antibody may be a full length antibody (e.g. having an intacthuman Fc region) or an antibody fragment (e.g. a Fab, Fab′ or F(ab′)₂).Furthermore, the antibody may be labeled with a detectable label,immobilized on a solid phase and/or conjugated with a heterologouscompound (such as a cytotoxic agent).

Diagnostic and therapeutic uses for the antibody are contemplated. Inone diagnostic application, the invention provides a method fordetermining the presence of VEGF protein comprising exposing a samplesuspected of containing the VEGF protein to the anti-VEGF antibody anddetermining binding of the antibody to the sample. For this use, theinvention provides a kit comprising the antibody and instructions forusing the antibody to detect the VEGF protein.

The invention further provides: isolated nucleic acid encoding theantibody; a vector comprising that nucleic acid, optionally operablylinked to control sequences recognized by a host cell transformed withthe vector; a host cell comprising that vector; a process for producingthe antibody comprising culturing the host cell so that the nucleic acidis expressed and, optionally, recovering the antibody from the host cellculture (e.g. from the host cell culture medium). The invention alsoprovides a composition comprising the anti-VEGF antibody and apharmaceutically acceptable carrier or diluent. The composition fortherapeutic use is sterile and may be lyophilized. The invention furtherprovides a method for treating a mammal suffering from a tumor orretinal disorder, comprising administering a therapeutically effectiveamount of the anti-VEGF antibody to the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the amino acid sequences of variable heavy domain(SEQ ID NO:9) and light domain (SEQ ID NO:10) of muMAbVEGF A.4.6.1,variable heavy domain (SEQ ID NO:7) and light domain (SEQ ID NO:8) ofhumanized F(ab) (F(ab)-12) and human consensus frameworks (hum III forheavy subgroup III (SEQ ID NO:11); humκ1 for light κ subgroup I (SEQ IDNO:12)). FIG. 1A aligns variable heavy domain sequences and FIG. 1Baligns variable light domain sequences. Asterisks indicate differencesbetween humanized F(ab)-12 and the murine MAb or between F(ab)-12 andthe human framework. Complementarity Determining Regions (CDRs) areunderlined.

FIG. 2 is a ribbon diagram of the model of humanized F(ab)-12 VL and VHdomains. VL domain is shown in brown with CDRs in tan. The sidechain ofresidue L46 is shown in yellow. VH domain is shown in purple with CDRsin pink. Sidechains of VH residues changed from human to murine areshown in yellow.

FIG. 3 depicts inhibition of VEGF-induced mitogenesis by humanizedanti-VEGF F(ab)-12 from Example 1. Bovine adrenal cortex-derivedcapillary endothelial cells were seeded at the density of 6×10³cells/well in six well plates, as described in Example 1. Either muMAbVEGF A.4.6.1 or rhuMAb VEGF (IgG1; F(ab)-12) was added at the indicatedconcentrations. After 2–3 hours, rhVEGF165 was added at the finalconcentration of 3 ng/ml. After five or six days, cells were trypsinizedand counted. Values shown are means of duplicate determinations. Thevariation from the mean did not exceed 10%.

FIG. 4 shows inhibition of tumor growth in vivo by humanized anti-VEGFF(ab)-12 from Example 1. A673 rhabdomyosarcoma cells were injected inBALB/c nude mice at the density of 2×10⁶ per mouse. Starting 24 hoursafter tumor cell inoculation, animals were injected with a control MAb,muMAb VEGF A4.6.1 or rhuVEGF MAb (IgG1; F(ab)-12) twice weekly, intraperitoneally. The dose of the control Mab was 5 mg/kg; the anti-VEGFMAbs were given at 0.5 or 5 mg/kg, as indicated (n=10). Four weeks aftertumor cell injection, animals were euthanized and tumors were removedand weighed. *: significant difference when compared to the controlgroup by ANOVA (p<0.05).

FIGS. 5A and 5B show the acid sequences of the light and heavy variabledomains respectively of murine antibody A4.6.1 (SEQ ID NO:10 for the VLand SEQ ID NO:9 for the VH) and humanized A4.6.1 variants hu2.0 (SEQ IDNO:13 for the VL and SEQ ID NO:14 for the VH) and hu2.10 (SEQ ID NO:15for the VL and SEQ ID NO:16 for the VH) from Example 2. Sequencenumbering is according to Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and mismatches are indicatedby asterisks (murine A4.6.1 vs hu2.0) or bullets (hu2.0 vs hu2.10).Variant hu2.0 contains only the CDR sequences (bold) from the murineantibody grafted onto a human light chain κ subgroup I consensusframework (SEQ ID NO:12) and heavy chain subgroup III consensusframework (SEQ ID NO:11). hu2.10 was the consensus humanized cloneobtained from phage sorting experiments described herein.

FIG. 6 depicts framework residues targeted for randomization in Example2.

FIG. 7 depicts the phagemid construct for surface display of Fab-pIIIfusions on phage. The phagemid encodes a humanized version of the Fabfragment for antibody A4.6.1 fused to a portion of the M13 gene III coatprotein. The fusion protein consists of the Fab joined at the carboxylterminus of the heavy chain to a single glutamine residue (fromsuppression of an amber codon in supE E. coli), then the C-terminalregion of the gene III protein (residues 249–406). Transformation intoF⁺ E. coli, followed by superinfection with M13KO7 helper phage,produces phagemid particles in which a small proportion of these displaya single copy of the fusion protein.

FIGS. 8A–E depict the double stranded nucleotide sequence (SEQ ID NO:99)for phage-display antibody vector phMB4-19-1.6 in Example 3 and theamino acid sequenceS encoded thereby (SEQ ID NOS: 100–102 and 130).

FIGS. 9A and 9B depict an alignment of the amino acid sequences for thelight and heavy variable domains respectively of affinity maturedanti-VEGF variants in Example 3, compared to F(ab)-12 of Example 1 (SEQID NO:8 and bases 1–118 of SEQ ID NO:7 for light and heavy variabledomains, respectively). CDRs are underlined and designated by L, light,or H, heavy chain, and numbers 1–3. Residues are numbered sequentiallyin the VL and VH domains, as opposed to the Kabat numbering scheme. Thetemplate molecule, MB1.6 (SEQ ID NOS: 103 and 104 for light and heavyvariable domains, respectively) is shown, along with variants: H2305.6(SEQ ID NOS: 105 and 106 for light and heavy variable domains,respectively), Y0101 (SEQ ID NOS: 107 and 108 for light and heavyvariable domains, respectively), and Y0192 (SEQ ID NOS: 109 and 110 forlight and heavy variable domains, respectively). Differences fromF(ab)-12 are shown in shaded boxes.

FIGS. 10A and 10B depict an alignment of the amino acid sequences forthe light and heavy variable domains respectively of affinity maturedanti-VEGF variants from Example 3 compared to F(ab)-12 of Example 1 (SEQID NO's 8 and 7 for light and heavy variable domains, respectively).CDRs are underlined and designated by L, light, or H, heavy chain, andnumbers 1–3. The variants are designated Y0243-1 (SEQ ID NOS: 111 and112 for light and heavy variable domains, respectively), Y0238-3 (SEQ IDNOS: 113 and 114 for light and heavy variable domains, respectively),Y0313-1 (SEQ ID NOS: 115 and 116 for light and heavy variable domains,respectively), and Y0317 (SEQ ID NOS: 117 and 118 for light and heavyvariable domains, respectively). Differences from F(ab)-12 are shown inshaded boxes.

FIG. 11 depicts the results of the HuVEC activity assay in Example 3 forvariants Y0238-3, Y0192 and Y0313-1 as well as full length F(ab)-12 fromExample 1.

FIG. 12 depicts inhibition of VEGF-induced mitogenesis by full lengthF(ab)-12 from Example 1 (rhuMAb VEGF), a Fab fragment of F(ab)-12 fromExample 1 (rhuFab VEGF), and a Fab fragment of affinity matured variantY0317 from Example 3 (rhuFab VEGF (affinity matured)).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The term “human VEGF” as used herein refers to the 165-amino acid humanvascular endothelial cell growth factor, and related 121-, 189-, and206-amino acid vascular endothelial cell growth factors, as described byLeung et al., Science 246:1306 (1989), and Houck et al., Mol. Endocrin.5:1806 (1991) together with the naturally occurring allelic andprocessed forms of those growth factors.

The present invention provides anti-VEGF antagonistic antibodies whichare capable of inhibiting one or more of the biological activities ofVEGF, for example, its mitogenic or angiogenic activity. Antagonists ofVEGF act by interfering with the binding of VEGF to a cellular receptor,by incapacitating or killing cells which have been activated by VEGF, orby interfering with vascular endothelial cell activation after VEGFbinding to a cellular receptor. All such points of intervention by aVEGF antagonist shall be considered equivalent for purposes of thisinvention.

The term “VEGF receptor” or “VEGFr” as used herein refers to a cellularreceptor for VEGF, ordinarily a cell-surface receptor found on vascularendothelial cells, as well as variants thereof which retain the abilityto bind hVEGF. One example of a VEGF receptor is the fms-like tyrosinekinase (flt), a transmembrane receptor in the tyrosine kinase family.DeVries et al., Science 255:989 (1992); Shibuya et al., Oncogene 5:519(1990). The flt receptor comprises an extracellular domain, atransmembrane domain, and an intracellular domain with tyrosine kinaseactivity. The extracellular domain is involved in the binding of VEGF,whereas the intracellular domain is involved in signal transduction.Another example of a VEGF receptor is the flk-1 receptor (also referredto as KDR). Matthews et al., Proc. Nat. Acad. Sci. 88:9026 (1991);Terman et al., Oncogene 6:1677 (1991); Terman et al., Biochem. Biophys.Res. Commun. 187:1579 (1992). Binding of VEGF to the flt receptorresults in the formation of at least two high molecular weightcomplexes, having apparent molecular weight of 205,000 and 300,000Daltons. The 300,000 Dalton complex is believed to be a dimer comprisingtwo receptor molecules bound to a single molecule of VEGF.

The term “epitope A4.6.1” when used herein, unless indicated otherwise,refers to the region of human VEGF to which the A4.6.1 antibodydisclosed in Kim et al., “Growth Factors 7:53 (1992) and Kim et al.Nature 362:841 (1993), binds.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework region (FR). The variabledomains of native heavy and light chains each comprise four FRs (FR1,FR2, FR3 and FR4, respectively), largely adopting a β-sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991), pages 647–669). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24–34 (L1),50–56 (L2) and 89–97 (L3) in the light chain variable domain and 31–35(H1), 50–65 (H2) and 95–102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26–32 (L1), 50–52 (L2) and 91–96 (L3) in the light chain variable domainand 26–32 (H1), 53–55 (H2) and 96–101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901–917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)–V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624–628 (1991) and Marks et al., J. Mol.Biol. 222:581–597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851–6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522–525 (1986); Reichmannet al., Nature 332:323–329 (1988); and Presta, Curr. Op. Struct. Biol.2:593–596 (1992).

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269–315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444–6448 (1993).

The expression “linear antibodies” when used throughout this applicationrefers to the antibodies described in Zapata et al. Protein Eng.8(10):1057–1062 (1995). Briefly, these antibodies comprise a pair oftandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

A “variant” anti-VEGF antibody, refers herein to a molecule whichdiffers in amino acid sequence from a “parent” anti-VEGF antibody aminoacid sequence by virtue of addition, deletion and/or substitution of oneor more amino acid residue(s) in the parent antibody sequence. In thepreferred embodiment, the variant comprises one or more amino acidsubstitution(s) in one or more hypervariable region(s) of the parentantibody. For example, the variant may comprise at least one, e.g. fromabout one to about ten, and preferably from about two to about five,substitutions in one or more hypervariable regions of the parentantibody. Ordinarily, the variant will have an amino acid sequencehaving at least 75% amino acid sequence identity with the parentantibody heavy or light chain variable domain sequences (e.g. as in SEQID NO:7 or 8), more preferably at least 80%, more preferably at least85%, more preferably at least 90%, and most preferably at least 95%.Identity or homology with respect to this sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical with the parent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. None of N-terminal, C-terminal, or internalextensions, deletions, or insertions into the antibody sequence shall beconstrued as affecting sequence identity or homology. The variantretains the ability to bind human VEGF and preferably has propertieswhich are superior to those of the parent antibody. For example, thevariant may have a stronger binding affinity, enhanced ability toinhibit VEGF-induced proliferation of endothelial cells and/or increasedability to inhibit VEGF-induced angiogenesis in vivo. To analyze suchproperties, one should compare a Fab form of the variant to a Fab formof the parent antibody or a full length form of the variant to a fulllength form of the parent antibody, for example, since it has been foundthat the format of the anti-VEGF antibody impacts its activity in thebiological activity assays disclosed herein. The variant antibody ofparticular interest herein is one which displays at least about 10 fold,preferably at least about 20 fold, and most preferably at least about 50fold, enhancement in biological activity when compared to the parentantibody.

The “parent” antibody herein is one which is encoded by an amino acidsequence used for the preparation of the variant. Preferably, the parentantibody has a human framework region and, if present, has humanantibody constant region(s). For example, the parent antibody may be ahumanized or human antibody.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The term “epitope tagged” when used herein refers to the anti-VEGFantibody fused to an “epitope tag”. The epitope tag polypeptide hasenough residues to provide an epitope against which an antibodythereagainst can be made, yet is short enough such that it does notinterfere with activity of the VEGF antibody. The epitope tag preferablyis sufficiently unique so that the antibody thereagainst does notsubstantially cross-react with other epitopes. Suitable tag polypeptidesgenerally have at least 6 amino acid residues and usually between about8–50 amino acid residues (preferably between about 9–30 residues).Examples include the flu HA tag polypeptide and its antibody 12CA5(Field et al. Mol. Cell. Biol. 8:2159–2165 (1988)); the c-myc tag andthe 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al.,Mol. Cell. Biol. 5(12):3610–3616 (1985)); and the Herpes Simplex virusglycoprotein D (gD) tag and its antibody (Paborsky et al., ProteinEngineering 3(6):547–553 (1990)). In certain embodiments, the epitopetag is a “salvage receptor binding epitope”. As used herein, the term“salvage receptor binding epitope” refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeAdriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (“Ara-C”),Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin,Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine,Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin,Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see U.S. Pat. No.4,675,187), Melphalan and other related nitrogen mustards.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375–382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery,” Borchardt et al., (ed.), pp.247–267, Humana Press (1985). The prodrugs of this invention include,but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibody.The label may itself be detectable by itself (e.g., radioisotope labelsor fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g. an affinity chromatography column). This term also includesa discontinuous solid phase of discrete particles, such as thosedescribed in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the anti-VEGF antibodies disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. An “isolated” nucleic acid moleculeis a nucleic acid molecule that is identified and separated from atleast one contaminant nucleic acid molecule with which it is ordinarilyassociated in the natural source of the antibody nucleic acid. Anisolated nucleic acid molecule is other than in the form or setting inwhich it is found in nature. Isolated nucleic acid molecules thereforeare distinguished from the nucleic acid molecule as it exists in naturalcells. However, an isolated nucleic acid molecule includes a nucleicacid molecule contained in cells that ordinarily express the antibodywhere, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

II. Modes for Carrying Out the Invention

The examples hereinbelow describe the production of humanized andvariant anti-VEGF antibodies with desirable properties from atherapeutic perspective including: (a) strong binding affinity for theVEGF antigen; (b) an ability to inhibit VEGF-induced proliferation ofendothelial cells in vitro; and (c) the ability to inhibit VEGF-inducedangiogenesis in vivo.

Antibody affinities may be determined as described in the exampleshereinbelow. Preferred humanized or variant antibodies are those whichbind human VEGF with a K_(d) value of no more than about 1×10⁻⁷M;preferably no more than about 1×10⁻⁸M; and most preferably no more thanabout 5×10⁻⁹M.

Aside from antibodies with strong binding affinity for human VEGF, it isalso desirable to select humanized or variant antibodies which haveother beneficial properties from a therapeutic perspective. For example,the antibody may be one which inhibits endothelial cell growth inresponse to VEGF. In one embodiment, the antibody may be able to inhibitbovine capillary endothelial cell proliferation in response to a nearmaximally effective concentration of VEGF (3 ng/ml). Preferably, theantibody has an effective dose 50 (ED50) value of no more than about 5nM, preferably no more than about 1 nM, and most preferably no more thanabout 0.5 nM, for inhibiting VEGF-induced proliferation of endothelialcells in this “endothelial cell growth assay”, i.e., at theseconcentrations the antibody is able to inhibit VEGF-induced endothelialcell growth in vitro by 50%. A preferred “endothelial cell growth assay”involves culturing bovine adrenal cortex-derived capillary endothelialcells in the presence of low glucose Dulbecco's modified Eagle's medium(DMEM) (GIBCO) supplemented with 10% calf serum, 2 mM glutamine, andantibiotics (growth medium), essentially as described in Example 1below. These endothelial cells are seeded at a density of 6×10³ cellsper well, in 6-well plates in growth medium. Either parent anti-VEGFantibody (control), humanized or variant anti-VEGF antibody is thenadded at concentrations ranging between 1 and 5000 ng/ml. After 2–3 hr,purified VEGF was added to a final concentration of 3 ng/ml. Forspecificity control, each antibody may be added to endothelial cells atthe concentration of 5000 ng/ml, either alone or in the presence of 2ng/ml bFGF. After five or six days, cells are dissociated by exposure totrypsin and counted in a Coulter counter (Coulter Electronics, Hialeah,Fla.). Data may be analyzed by a four-parameter curve fitting program(KaleidaGraph).

The preferred humanized or variant anti-VEGF antibody may also be onewhich has in vivo tumor suppression activity. For example, the antibodymay suppress the growth of human A673 rhabdomyosarcoma cells or breastcarcinoma MDA-MB-435 cells in nude mice. For in vivo tumor studies,human A673 rhabdomyosarcoma cells (ATCC; CRL 1598) or MDA-MB-435 cells(available from the ATCC) are cultured in DMEM/F12 supplemented with 10%fetal bovine serum, 2 mM glutamine and antibiotics as described inExample 1 below. Female BALB/c nude mice, 6–10 weeks old, are injectedsubcutaneously with 2×10⁶ tumor cells in the dorsal area in a volume of200 μl. Animals are then treated with the humanized or variant antibodyand a control antibody with no activity in this assay. The humanized orvariant anti-VEGF MAb is administered at a dose of 0.5 and/or 5 mg/kg.Each MAb is administered twice weekly intra peritoneally in a volume of100 μl, starting 24 hr after tumor cell inoculation. Tumor size isdetermined at weekly intervals. Four weeks after tumor cell inoculation,animals are euthanized and the tumors are removed and weighed.Statistical analysis may be performed by ANOVA. Preferably, the antibodyin this “in vivo tumor assay” inhibits about 50–100%, preferably about70–100% and most preferably about 80–100% human A673 tumor cell growthat a dose of 5 mg/kg.

In the preferred embodiment, the humanized or variant antibody fails toelicit an immunogenic response upon administration of a therapeuticallyeffective amount of the antibody to a human patient. If an immunogenicresponse is elicited, preferably the response will be such that theantibody still provides a therapeutic benefit to the patient treatedtherewith.

The humanized or variant antibody is also preferably one which is ableto inhibit VEGF-induced angiogenesis in a human, e.g. to inhibit humantumor growth and/or inhibit intraocular angiogenesis in retinaldisorders.

Preferred antibodies bind the “epitope A4.6.1” as herein defined. Toscreen for antibodies which bind to the epitope on human VEGF bound byan antibody of interest (e.g., those which block binding of the A4.6.1antibody to human VEGF), a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al., J.Biol. Chem. 270:1388–1394 (1995), can be performed to determine whetherthe antibody binds an epitope of interest.

The antibodies of the preferred embodiment herein have a heavy chainvariable domain comprising an amino acid sequence represented by theformula: FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4, wherein “FR1–4” representthe four framework regions and “CDRH1–3” represent the threehypervariable regions of an anti-VEGF antibody variable heavy domain.FR1–4 may be derived from a “consensus sequence” (i.e. the most commonamino acids of a class, subclass or subgroup of heavy or light chains ofhuman immunoglobulins) as in the examples below or may be derived froman individual human antibody framework region or from a combination ofdifferent framework region sequences. Many human antibody frameworkregion sequences are compiled in Kabat et al., supra, for example. Inone preferred embodiment, the variable heavy FR is provided by aconsensus sequence of a human immunoglobulin subgroup as compiled byKabat et al., supra. Preferably, the human immunoglobulin subgroup ishuman heavy chains subgroup III (e.g. as in SEQ ID NO:11).

The human variable heavy FR sequence preferably has substitutionstherein, e.g. wherein the human FR residue is replaced by acorresponding nonhuman residue (by “corresponding nonhuman residue” ismeant the nonhuman residue with the same Kabat positional numbering asthe human residue of interest when the human and nonhuman sequences arealigned), but replacement with the nonhuman residue is not necessary.For example, a replacement FR residue other than the correspondingnonhuman residue may be selected by phage display (see Example 2 below).Exemplary variable heavy FR residues which may be substituted includeany one or more of FR residue numbers: 37H, 49H, 67H, 69H, 71 H, 73H,75H, 76H, 78H, 94H (Kabat residue numbering employed here). Preferablyat least two, or at least three, or at least four of these residues aresubstituted. A particularly preferred combination of FR substitutionsis: 49H, 69H, 71H, 73H, 76H, 78H, and 94H.

With respect to the heavy chain hypervariable regions, these preferablyhave amino acid sequences as follows:

CDRH1

-   GYX₁X₂X₃X₄YGX₅N (SEQ ID NO:119), wherein X₁ is D, T or E, but    preferably is D or T; X₂ is F, W, or Y, but preferably is F; X₃ is    T, Q, G or S, but preferably is T; X₄ is H or N; and X₅ is M or I,    but preferably is M.    CDRH2-   WINTX₁TGEPTYAADFKR (SEQ ID NO:120), wherein X₁ is Y or W, but    preferably is Y.    CDRH3-   YPX₁YX₂X₃X₄X₅HWYFDV (SEQ ID NO:121), wherein X₁ is H or Y; X₂ is Y,    R, K, I, T, E, or W, but preferably is Y; X₃ is G, N, A, D, Q, E, T,    K, or S, but preferably is G; X₄ is S, T, K, Q, N, R, A, E, or G,    but preferably is S or T; and X₅ is S or G, but preferably is S.

The heavy chain variable domain optionally comprises what has beendesignated “CDR7” herein within (i.e. forming part of) FR3 (see FIGS. 9Band 10B), wherein CDR7 may have the following amino acid sequence:

CDR7

-   X₁SX₂DX₃X₄X₅X₆TX₇ (SEQ ID NO:122), wherein X₁ is F, I , V, L, or A,    but preferably is F; X₂ is A, L, V, or I, but preferably is L; X₃ is    T, V or K, but preferably is T; X₄ is S or W, but preferably is S;    X₅ is S, or K, but preferably is K; X₆ is N, or S, but preferably is    S; and X₇ is V, A, L or I, but preferably is A.

The antibodies of the preferred embodiment herein have a light chainvariable domain comprising an amino acid sequence represented by theformula: FR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4, wherein “FR1–4” representthe four framework regions and “CDRL1-3” represent the threehypervariable regions of an anti-VEGF antibody variable heavy domain.FR1-4 may be derived from a “consensus sequence” (i.e. the most commonamino acids of a class, subclass or subgroup of heavy or light chains ofhuman immunoglobulins) as in the examples below or may be derived froman individual human antibody framework region or from a combination ofdifferent framework region sequences. In one preferred embodiment, thevariable light FR is provided by a consensus sequence of a humanimmunoglobulin subgroup as compiled by Kabat et al., supra. Preferably,the human immunoglobulin subgroup is human kappa light chains subgroup I(e.g. as in SEQ ID NO:12).

The human variable light FR sequence preferably has substitutionstherein, e.g. wherein the human FR residue is replaced by acorresponding mouse residue, but replacement with the nonhuman residueis not necessary. For example, a replacement residue other than thecorresponding nonhuman residue may be selected by phage display (seeExample 2 below). Exemplary variable light FR residues which may besubstituted include any one or more of FR residue numbers: 4L, 46L and71L (Kabat residue numbering employed here). Preferably only 46L issubstituted. In another embodiment, both 4L and 46L are substituted.

With respect to the CDRs, these preferably have amino acid sequences asfollows:

CDRL1

-   X₁AX₂X₃X₄X₅SNYLN (SEQ ID NO:123), wherein X₁ is R or S, but    preferably is S; X₂ is S or N, but preferably is S; X₃ is Q or E,    but preferably is Q; X₄ is Q or D, but preferably is D; and X₅ is I    or L, but preferably is I.    CDRL2-   FTSSLHS (SEQ ID NO:124).    CDRL3-   QQYSX₁X₂PWT (SEQ ID NO:125), wherein X₁ is T, A or N, but preferably    is T; and X₂ is V or T, but preferably is V.

Preferred humanized anti-VEGF antibodies are those having the heavyand/or light variable domain sequences of F(ab)-12 in Example 1 andvariants thereof such as affinity matured forms including variantsY0317, Y0313-1 and Y0238-3 in Example 3, with Y0317 being the preferredvariant. Methods for generating humanized anti-VEGF antibodies ofinterest herein are elaborated in more detail below.

A. Antibody Preparation

Methods for humanizing nonhuman VEGF antibodies and generating variantsof anti-VEGF antibodies are described in the examples below. In order tohumanize an anti-VEGF antibody, the nonhuman antibody starting materialis prepared. Where a variant is to be generated, the parent antibody isprepared. Exemplary techniques for generating such nonhuman antibodystarting material and parent antibodies will be described in thefollowing sections.

(i) Antigen Preparation

The VEGF antigen to be used for production of antibodies may be, e.g.,intact VEGF or a fragment of VEGF (e.g. a VEGF fragment comprising“epitope A4.6.1”). Other forms of VEGF useful for generating antibodieswill be apparent to those skilled in the art. The VEGF antigen used togenerate the antibody, is preferably human VEGF, e.g. as described inLeung et al., Science 246:1306 (1989), and Houck et al., Mol. Endocrin.5:1806 (1991).

(ii) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(iii) Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59–103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOP-21 and M.C.-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51–63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59–103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Recombinant production of antibodies will be described in moredetail below.

(iv) Humanization and Amino Acid Sequence Variants

Examples 1–2 below describe procedures for humanization of an anti-VEGFantibody. In certain embodiments, it may be desirable to generate aminoacid sequence variants of these humanized antibodies, particularly wherethese improve the binding affinity or other biological properties of thehumanized antibody. Example 3 describes methodologies for generatingamino acid sequence variants of an anti-VEGF antibody with enhancedaffinity relative to the parent antibody.

Amino acid sequence variants of the anti-VEGF antibody are prepared byintroducing appropriate nucleotide changes into the anti-VEGF antibodyDNA, or by peptide synthesis. Such variants include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the anti-VEGF antibodies of theexamples herein. Any combination of deletion, insertion, andsubstitution is made to arrive at the final construct, provided that thefinal construct possesses the desired characteristics. The amino acidchanges also may alter post-translational processes of the humanized orvariant anti-VEGF antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theanti-VEGF antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis,” as described by Cunningham andWells Science, 244:1081–1085 (1989). Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with VEGF antigen. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed anti-VEGFantibody variants are screened for the desired activity. Alaninescanning mutagenesis is described in Example 3.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-VEGF antibody with an N-terminal methionyl residue or theantibody fused to an epitope tag. Other insertional variants of theanti-VEGF antibody molecule include the fusion to the N- or C-terminusof the anti-VEGF antibody of an enzyme or a polypeptide which increasesthe serum half-life of the antibody (see below).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the anti-VEGF antibodymolecule removed and a different residue inserted in its place. Thesites of greatest interest for substitutional mutagenesis include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;asp, lys; gln arg Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn;glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H) asn; gln; lys; argarg Ile (I) leu; val; met; ala; leu phe; norleucine Leu (L) norleucine;ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe;ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thrthr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser pheVal (V) ile; leu; met; phe; leu ala; norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the humanized or variant anti-VEGF antibody also may be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)may be added to the antibody to improve its stability (particularlywhere the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants is affinity maturation using phage display (see Example 3herein). Briefly, several hypervariable region sites (e.g. 6–7 sites)are mutated to generate all possible amino substitutions at each site.The antibody variants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants are then screened for their biological activity (e.g. bindingaffinity) as herein disclosed. In order to identify candidatehypervariable region sites for modification, alanine scanningmutagenesis (see Example 3) can be performed to identified hypervariableregion residues contributing significantly to antigen binding.Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and human VEGF. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-VEGF antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the anti-VEGF antibody.

(v) Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255–258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669,5,589,369 and 5,545,807. Human antibodies can also be derived fromphage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol, 222:581–597 (1991); and U.S. Pat.Nos. 5,565,332 and 5,573,905). As discussed above, human antibodies mayalso be generated by in vitro activated B cells (see U.S. Pat. Nos.5,567,610 and 5,229,275)

(vi) Antibody Fragments

In certain embodiments, the humanized or variant anti-VEGF antibody isan antibody fragment. Various techniques have been developed for theproduction of antibody fragments. Traditionally, these fragments werederived via proteolytic digestion of intact antibodies (see, e.g.,Morimoto et al., Journal of Biochemical and Biophysical Methods24:107–117 (1992) and Brennan et al., Science 229:81 (1985)). However,these fragments can now be produced directly by recombinant host cells.For example, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163–167 (1992)). In another embodiment, the F(ab′)₂ isformed using the leucine zipper GCN4 to promote assembly of the F(ab′)₂molecule. According to another approach, Fv, Fab or F(ab′)₂ fragmentscan be isolated directly from recombinant host cell culture. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner.

(vii) Multispecific Antibodies

In some embodiments, it may be desirable to generate multispecific (e.g.bispecific) humanized or variant anti-VEGF antibodies having bindingspecificities for at least two different epitopes. Exemplary bispecificantibodies may bind to two different epitopes of the VEGF protein.Alternatively, an anti-VEGF arm may be combined with an arm which bindsto a triggering molecule on a leukocyte such as a T-cell receptormolecule (e.g., CD2 or CD3), or Fc receptors for IgG (FcγR), such asFcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellulardefense mechanisms to the VEGF-expressing cell. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which expressVEGF. These antibodies possess an VEGF-binding arm and an arm whichbinds the cytotoxic agent (e.g., saporin, anti-interferon-60, vincaalkaloid, ricin A chain, methotrexate or radioactive isotope hapten).Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g., F(ab′)₂ bispecific antibodies).

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.,tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. See WO96/27011 published Sep. 6, 1996.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes. In yet afurther embodiment, Fab′-SH fragments directly recovered from E. colican be chemically coupled in vitro to form bispecific antibodies.Shalaby et al., J. Exp. Med. 175:217–225 (1992).

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547–1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444–6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol. 152:5368 (1994). Alternatively,the bispecific antibody may be a “linear antibody” produced as describedin Zapata et al. Protein Eng. 8(10):1057–1062 (1995).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

(viii) Other Modifications

Other modifications of the humanized or variant anti-VEGF antibody arecontemplated. For example, it may be desirable to modify the antibody ofthe invention with respect to effector function, so as to enhance theeffectiveness of the antibody in treating cancer, for example. Forexample cysteine residue(s) may be introduced in the Fc region, therebyallowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved internalizationcapability and/or increased complement-mediated cell killing andantibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J.Exp Med. 176:1191–1195 (1992) and Shopes, B. J. Immunol. 148:2918–2922(1992). Homodimeric antibodies with enhanced anti-tumor activity mayalso be prepared using heterobifunctional cross-linkers as described inWolff et al., Cancer Research 53:2560–2565 (1993). Alternatively, anantibody can be engineered which has dual Fc regions and may therebyhave enhanced complement lysis and ADCC capabilities. See Stevenson etal., Anti-Cancer Drug Design 3:219–230 (1989).

The invention also pertains to immunoconjugates comprising the antibodydescribed herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated anti-VEGF antibodies. Examples include ²¹²Bi, ¹³¹I,¹³¹In, ⁹⁰Y and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) which is conjugatedto a cytotoxic agent (e.g., a radionuclide).

The anti-VEGF antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci.USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286–288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19):1984 (1989)

The antibody of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature328:457–458 (1987)). Antibody-abzyme conjugates can be prepared asdescribed herein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-VEGFantibodies by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature 312:604–608 (1984)).

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. In this case, it may be desirable to modifythe antibody fragment in order to increase its serum half life. This maybe achieved, for example, by incorporation of a salvage receptor bindingepitope into the antibody fragment (e.g., by mutation of the appropriateregion in the antibody fragment or by incorporating the epitope into apeptide tag that is then fused to the antibody fragment at either end orin the middle, e.g., by DNA or peptide synthesis). See WO96/32478published Oct. 17, 1996.

The salvage receptor binding epitope generally constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the antibody fragment.

In one most preferred embodiment, the salvage receptor binding epitopecomprises the sequence: PKNSSMISNTP (SEQ ID NO:17), and optionallyfurther comprises a sequence selected from the group consisting ofHQSLGTQ (SEQ ID NO:18), HQNLSDGK (SEQ ID NO:19), HQNISDGK (SEQ IDNO:20), or VISSHLGQ (SEQ ID NO:21), particularly where the antibodyfragment is a Fab or F(ab′)₂. In another most preferred embodiment, thesalvage receptor binding epitope is a polypeptide containing thesequence(s): HQNLSDGK (SEQ ID NO:19), HQNISDGK (SEQ ID NO:20), orVISSHLGQ (SEQ ID NO:21) and the sequence: PKNSSMISNTP (SEQ ID NO:17).

Covalent modifications of the humanized or variant anti-VEGF antibodyare also included within the scope of this invention. They may be madeby chemical synthesis or by enzymatic or chemical cleavage of theantibody, if applicable. Other types of covalent modifications of theantibody are introduced into the molecule by reacting targeted aminoacid residues of the antibody with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues. Exemplary covalent modifications of polypeptides are describedin U.S. Pat. No. 5,534,615, specifically incorporated herein byreference. A preferred type of covalent modification of the antibodycomprises linking the antibody to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

B. Vectors, Host Cells and Recombinant Methods

The invention also provides isolated nucleic acid encoding the humanizedor variant anti-VEGF antibody, vectors and host cells comprising thenucleic acid, and recombinant techniques for the production of theantibody.

For recombinant production of the antibody, the nucleic acid encoding itmay be isolated and inserted into a replicable vector for furthercloning (amplification of the DNA) or for expression. In anotherembodiment, the antibody may be produced by homologous recombination,e.g. as described in U.S. Pat. No. 5,204,244, specifically incorporatedherein by reference. DNA encoding the monoclonal antibody is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence, e.g., as described in U.S.Pat. No. 5,534,615 issued Jul. 9, 1996 and specifically incorporatedherein by reference.

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-VEGFantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-VEGFantibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa califonica NPV and the Bm-5 strain of Bombyx mori NPV, andsuch viruses may be used as the virus herein according to the presentinvention, particularly for transfection of Spodoptera frugiperda cells.Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243–251(1980) ); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44–68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-VEGF antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

The host cells used to produce the anti-VEGF antibody of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing the host cells. In addition, any ofthe media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes etal., Anal. Biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S.Pat. No. Re. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163–167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1–13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5–4.5, preferably performed at low salt concentrations(e.g.,from about 0–0.25M salt).

C. Pharmaceutical Formulations

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other(see Section F below). Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

D. Non-therapeutic Uses for the Antibody

The antibodies of the invention may be used as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a Sephadex resin or filter paper, using methods well known in theart. The immobilized antibody is contacted with a sample containing theVEGF protein (or fragment thereof) to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the VEGF protein, which is boundto the immobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, that will release theVEGF protein from the antibody.

Anti-VEGF antibodies may also be useful in diagnostic assays for VEGFprotein, e.g., detecting its expression in specific cells, tissues, orserum. Such diagnostic methods may be useful in cancer diagnosis.

For diagnostic applications, the antibody typically will be labeled witha detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodycan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed.Wiley-Interscience, New York, N.Y., Pubs. (1991) for example andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147–166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g., digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g., anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the anti-VEGF antibody need notbe labeled, and the presence thereof can be detected using a labeledantibody which binds to the VEGF antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147–158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of VEGF protein in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radio nuclide (such as ¹¹¹In,⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

E. Diagnostic Kits

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labeled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

F. Therapeutic Uses for the Antibody

For therapeutic applications, the anti-VEGF antibodies of the inventionare administered to a mammal, preferably a human, in a pharmaceuticallyacceptable dosage form such as those discussed above, including thosethat may be administered to a human intravenously as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes. Theantibodies also are suitably administered by intra tumoral, peritumoral,intralesional, or perilesional routes, to exert local as well assystemic therapeutic effects. The intraperitoneal route is expected tobe particularly useful, for example, in the treatment of ovarian tumors.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

The anti-VEGF antibodies are useful in the treatment of variousneoplastic and non-neoplastic diseases and disorders. Neoplasms andrelated conditions that are amenable to treatment include breastcarcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas,colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas,arrhenoblastomas, cervical carcinomas, endometrial carcinoma,endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma,head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas,hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma,cavernous hemangioma, hemangioblastoma, pancreas carcinomas,retinoblastoma, astrocytoma, glioblastoma, Schwannoma,oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroidcarcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma,abnormal vascular proliferation associated with phakomatoses, edema(such as that associated with brain tumors), and Meigs' syndrome.

Non-neoplastic conditions that are amenable to treatment includerheumatoid arthritis, psoriasis, atherosclerosis, diabetic and otherproliferative retinopathies including retinopathy of prematurity,retrolental fibroplasia, neovascular glaucoma, age-related maculardegeneration, thyroid hyperplasias (including Grave's disease), cornealand other tissue transplantation, chronic inflammation, lunginflammation, nephrotic syndrome, preeclampsia, ascites, pericardialeffusion (such as that associated with pericarditis), and pleuraleffusion.

Age-related macular degeneration (AMD) is a leading cause of severevisual loss in the elderly population. The exudative form of AMD ischaracterized by choroidal neovascularization and retinal pigmentepithelial cell detachment. Because choroidal neovascularization isassociated with a dramatic worsening in prognosis, the VEGF antibodys ofthe present invention are expected to be especially useful in reducingthe severity of AMD.

Depending on the type and severity of the disease, about 1 μg/kg toabout 50 mg/kg (e.g., 0.1–20 mg/kg) of antibody is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily or weekly dosage might range from about 1 μg/kg to about 20 mg/kgor more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is repeated until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays, including, for example, radiographic tumor imaging.

According to another embodiment of the invention, the effectiveness ofthe antibody in preventing or treating disease may be improved byadministering the antibody serially or in combination with another agentthat is effective for those purposes, such as tumor necrosis factor(TNF), an antibody capable of inhibiting or neutralizing the angiogenicactivity of acidic or basic fibroblast growth factor (FGF) or hepatocytegrowth factor (HGF), an antibody capable of inhibiting or neutralizingthe coagulant activities of tissue factor, protein C, or protein S (seeEsmon et al., PCT Patent Publication No. WO 91/01753, published 21 Feb.1991), an antibody capable of binding to HER2 receptor (see Hudziak etal., PCT Patent Publication No. WO 89/06692, published 27 Jul. 1989), orone or more conventional therapeutic agents such as, for example,alkylating agents, folic acid antagonists, anti-metabolites of nucleicacid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil,cisplatin, purine nucleosides, amines, amino acids, triazol nucleosides,or corticosteroids. Such other agents may be present in the compositionbeing administered or may be administered separately. Also, the antibodyis suitably administered serially or in combination with radiologicaltreatments, whether involving irradiation or administration ofradioactive substances.

In one embodiment, vascularization of tumors is attacked in combinationtherapy. The antibody and one or more other anti-VEGF antagonists areadministered to tumor-bearing patients at therapeutically effectivedoses as determined for example by observing necrosis of the tumor orits metastatic foci, if any. This therapy is continued until such timeas no further beneficial effect is observed or clinical examinationshows no trace of the tumor or any metastatic foci. Then TNF isadministered, alone or in combination with an auxiliary agent such asalpha-, beta-, or gamma-interferon, anti-HER2 antibody, heregulin,anti-heregulin antibody, D-factor, interleukin-1 (IL-1), interleukin-2(IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), oragents that promote microvascular coagulation in tumors, such asanti-protein C antibody, anti-protein S antibody, or C4b binding protein(see Esmon et al., PCT Patent Publication No. WO 91/01753, published 21Feb. 1991), or heat or radiation.

Since the auxiliary agents will vary in their effectiveness it isdesirable to compare their impact on the tumor by matrix screening inconventional fashion. The administration of anti-VEGF antibody and TNFis repeated until the desired clinical effect is achieved.Alternatively, the anti-VEGF antibody is administered together with TNFand, optionally, auxiliary agent(s). In instances where solid tumors arefound in the limbs or in other locations susceptible to isolation fromthe general circulation, the therapeutic agents described herein areadministered to the isolated tumor or organ. In other embodiments, a FGFor platelet-derived growth factor (PDGF) antagonist, such as an anti-FGFor an anti-PDGF neutralizing antibody, is administered to the patient inconjunction with the anti-VEGF antibody. Treatment with anti-VEGFantibodies optimally may be suspended during periods of wound healing ordesirable neovascularization.

G. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from avariety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the anti-VEGF antibody.The label on, or associated with, the container indicates that thecomposition is used for treating the condition of choice. The article ofmanufacture may further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

EXAMPLE 1

This example describes the production of humanized anti-VEGF antibodieswith desirable properties from a therapeutic standpoint.

Materials and Methods

Cloning of Murine A4.6.1 MAb and Construction of Mouse-Human ChimericFab: The murine anti-VEGF mAb A4.6.1 has been previously described byKim et al., Growth Factors 7:53 (1992) and Kim et al. Nature 362:841(1993). Total RNA was isolated from hybridoma cells producing theanti-VEGF Mab A.4.6.1 using RNAsol (TEL-TEST) and reverse-transcribed tocDNA using Oligo-dT primer and the SuperScript II system (GIBCO BRL,Gaithersburg, Md.). Degenerate oligonucleotide primer pools, based ofthe N-terminal amino acid sequences of the light and heavy chains of theantibody, were synthesized and used as forward primers. Reverse primerswere based on framework 4 sequences obtained from murine light chainsubgroup kV and heavy chain subgroup II (Kabat et al. Sequences ofProteins of Immunological Interest. 5th ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). After polymerasechain reaction (PCR) amplification, DNA fragments were ligated to a TAcloning vector (Invitrogen, San Diego, Calif.). Eight clones each of thelight and heavy chains were sequenced. One clone with a consensussequence for the light chain VL domain and one with a consensus sequencefor the heavy chain VH domain were subcloned respectively into the pEMX1vector containing the human CL and CH1 domains (Werther et al. J.Immunol. 157:4986–4995 (1996)), thus generating a mouse-human chimera.This chimeric F(ab) consisted of the entire murine A4.6.1 VH domainfused to a human CH1 domain at amino acid SerH113 and the entire murineA4.6.1 VL domain fused to a human CL domain at amino acid LysL107.Expression and purification of the chimeric F(ab) were identical to thatof the humanized F(ab)s. The chimeric F(ab) was used as the standard inthe binding assays.

Computer Graphics Models of Murine and Humanized F(ab): Sequences of theVL and VH domains (FIGS. 1A and 1B) were used to construct a computergraphics model of the murine A4.6.1 VL-VH domains. This model was usedto determine which framework residues should be incorporated into thehumanized antibody. A model of the humanized F(ab) was also constructedto verify correct selection of murine framework residues. Constructionof models was performed as described previously (Carter et al. Proc.Natl. Acad. Sci. USA 89:4285–4289 (1992) and Eigenbrot et al. J. Mol.Biol. 229:969–995 (1993)).

Construction of Humanized F(ab)s: The plasmid pEMX1 used for mutagenesisand expression of F(ab)s in E. coli has been described previously(Werther et al., supra). Briefly, the plasmid contains a DNA fragmentencoding a consensus human κ subgroup I light chain (VLkI-CL) and aconsensus human subgroup III heavy chain (VHIII-CH1) and an alkalinephosphatase promoter. The use of the consensus sequences for VL and VHhas been described previously (Carter et al., supra).

To construct the first F(ab) variant of humanized A4.6.1, F(ab)-1,site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA82:488–492 (1985)) was performed on a deoxyuridine-containing templateof pEMX1. The six CDRs according to Kabat et al., supra, were changed tothe murine A4.6.1 sequence. F(ab)-1 therefore consisted of a completehuman framework (VL k subgroup I and VH subgroup III) with the sixcomplete murine CDR sequences. Plasmids for all other F(ab) variantswere constructed from the plasmid template of F(ab)-1. Plasmids weretransformed into E. coli strain XL-1 Blue (Stratagene, San Diego,Calif.) for preparation of double- and single-stranded DNA. For eachvariant, DNA coding for light and heavy chains was completely sequencedusing the dideoxynucleotide method (Sequenase, U.S. Biochemical Corp.,Cleveland, Ohio). Plasmids were transformed into E. coli strain 16C9, aderivative of MM294, plated onto Luria broth plates containing 50 μg/mlcarbenicillin, and a single colony selected for protein expression. Thesingle colony was grown in 5 ml Luria broth-100 mg/ml carbenicillin for5–8 h at 37° C. The 5 ml culture was added to 500 ml AP5–50 μg/mlcarbenicillin and allowed to grow for 20 h in a 4 L baffled shake flaskat 30° C. AP5 media consists of: 1.5 g glucose, 11.0 g Hycase SF, 0.6 gyeast extract (certified), 0.19 g MgSO4 (anhydrous), 1.07 g NH4Cl, 3.73g KCI, 1.2 g NaCl, 120 ml 1 M triethanolamine, pH 7.4, to 1 L water andthen sterile filtered through 0.1 mm Sealkeen filter. Cells wereharvested by centrifugation in a 1 L centrifuge bottle at 3000×g and thesupernatant removed. After freezing for 1 h, the pellet was resuspendedin 25 ml cold 10 mM Tris-1 mM EDTA-20% sucrose, pH 8.0. 250 ml of 0.1 Mbenzamidine (Sigma, St. Louis, Mo.) was added to inhibit proteolysis.After gentle stirring on ice for 3 h, the sample was centrifuged at40,000×g for 15 min. The supernatant was then applied to a proteinG-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) column (0.5 ml bedvolume) equilibrated with 10 mM Tris-1 mM EDTA, pH 7.5. The column waswashed with 10 ml of 10 mM Tris-1 mM EDTA, pH 7.5, and eluted with 3 ml0.3 M glycine, pH 3.0, into 1.25 ml 1 M Tris, pH 8.0. The F(ab) was thenbuffer exchanged into PBS using a Centricon-30 (Amicon, Beverly, Mass.)and concentrated to a final volume of 0.5 ml. SDS-PAGE gels of allF(ab)s were run to ascertain purity and the molecular weight of eachvariant was verified by electrospray mass spectrometry.

Construction and Expression of Chimeric and Humanized IgG: Forgeneration of human IgG1 variants of chimeric (chIgG1) and humanized(huIgG1) A4.6.1, the appropriate murine or humanized VL and VH(F(ab)-12, Table 2) domains were subcloned into separate, previouslydescribed, pRK vectors (Eaton et al., Biochemistry 25:8343–8347 (1986)).The DNA coding for the entire light and the entire heavy chain of eachvariant was verified by dideoxynucleotide sequencing.

For transient expression of variants, heavy and light chain plasmidswere co-transfected into human 293 cells (Graham et al., J. Gen. Virol.36:59–74 (1977)), using a high efficiency procedure (Gorman et al., DNAProt. Eng. Tech. 2:3–10 (1990)). Media was changed to serum-free andharvested daily for up to five days. Antibodies were purified from thepooled supernatants using protein A-Sepharose CL-4B (Pharmacia). Theeluted antibody was buffer exchanged into PBS using a Centricon-30(Amicon), concentrated to 0.5 ml, sterile filtered using a Millex-GV(Millipore, Bedford, Mass.) and stored at 4° C.

For stable expression of the final humanized IgG1 variant (rhuMAb VEGF),Chinese hamster ovary (CHO) cells were transfected with dicistronicvectors designed to coexpress both heavy and light chains (Lucas et al.,Nucleic Acid Res. 24:1774–79 (1996)). Plasmids were introduced into DP12cells, a proprietary derivative of the CHO-K1 DUX B11 cell linedeveloped by L. Chasin (Columbia University), via lipofection andselected for growth in GHT-free medium (Chisholm, V. High efficiencygene transfer in mammalian cells. In: Glover, D M, Hames, B D. DNACloning 4. Mammalian systems. Oxford Univ. Press, Oxford pp 1–41(1996)). Approximately 20 unamplified clones were randomly chosen andreseeded into 96 well plates. Relative specific productivity of eachcolony was monitored using an ELISA to quantitate the full length humanIgG accumulated in each well after 3 days and a fluorescent dye, CalcienAM, as a surrogate marker of viable cell number per well. Based on thesedata, several unamplified clones were chosen for further amplificationin the presence of increasing concentrations of methotrexate. Individualclones surviving at 10, 50, and 100 nM methotrexate were chosen andtransferred to 96 well plates for productivity screening. One clone,which reproducibly exhibited high specific productivity, was expanded inT-flasks and used to inoculate a spinner culture. After severalpassages, the suspension-adapted cells were used to inoculate productioncultures in GHT-containing, serum-free media supplemented with varioushormones and protein hydrolysates. Harvested cell culture fluidcontaining rhuMAb VEGF was purified using protein A-Sepharose CL-4B. Thepurity after this step was ˜99%. Subsequent purification to homogeneitywas carried out using an ion exchange chromatography step. The endotoxincontent of the final purified antibody was <0.10 eu/mg.

F(ab) and IgG Quantitation: For quantitating F(ab) molecules, ELISAplates were coated with 2 μg/ml goat anti-human IgG Fab (OrganonTeknika, Durham, N.C.) in 50 mM carbonate buffer, pH 9.6, at 4° C.overnight and blocked with PBS-0.5% bovine serum albumin (blockingbuffer) at room temperature for 1 h. Standards (0.78-50 ng/ml humanF(ab)) were purchased from Chemicon (Temecula, Calif.). Serial dilutionsof samples in PBS-0.5% bovine serum albumin-0.05% polysorbate 20 (assaybuffer) were incubated on the plates for 2 h. Bound F(ab) was detectedusing horseradish peroxidase-labeled goat anti-human IgG F(ab) (OrganonTeknika), followed by 3,3′,5,5′-tetramethylbenzidine (Kirkegaard & PerryLaboratories, Gaithersburg, Md.) as the substrate. Plates were washedbetween steps. Absorbance was read at 450 nm on a Vmax plate reader(Molecular Devices, Menlo Park, Calif.). The standard curve was fitusing a four-parameter nonlinear regression curve-fitting program. Datapoints which fell in the range of the standard curve were used forcalculating the F(ab) concentrations of samples. The concentration offull-length antibody was determined using goat anti-human IgG Fc(Cappel, Westchester, Pa.) for capture and horseradishperoxidase-labeled goat anti-human Fc (Cappel) for detection. Human IgG1(Chemicon) was used as standard.

VEGF Binding Assay: For measuring the VEGF binding activity of F(ab)s,ELISA plates were coated with 2 μg/ml rabbit F(ab′)₂ to human IgG Fc(Jackson ImmunoResearch, West Grove, Pa.) and blocked with blockingbuffer (described above). Diluted conditioned medium containing 3 ng/mlof KDR-lgG (Park et al., J. Biol. Chem. 269:25646–25645 (1994)) inblocking buffer were incubated on the plate for 1 h. Standards (6.9–440ng/ml chimeric F(ab)) and two-fold serial of samples were incubated with2 nM biotinylated VEGF for 1 h in tubes. The solutions from the tubeswere then transferred to the ELISA plates and incubated for 1 h. Afterwashing, biotinylated VEGF bound to KDR was detected using horseradishperoxidase-labeled streptavidin (Zymed, South San Francisco, Calif. orSigma, St. Louis, Mo.) followed by 3,3′,5,5′-tetramethylbenzidine as thesubstrate. Titration curves were fit with a four-parameter nonlinearregression curve-fitting program (KaleidaGraph, Synergy Software,Reading Pa.). Concentrations of F(ab) variants corresponding to themidpoint absorbance of the titration curve of the standard werecalculated and then divided by the concentration of the standardcorresponding to the midpoint absorbance of the standard titrationcurve. Assays for full-length IgG were the same as for the F(ab)s exceptthat the assay buffer contained 10% human serum.

BIAcore™ Biosensor Assay: VEGF binding of the humanized and chimericF(ab)s were compared using a BIAcore™ biosensor (Karlsson et al.Methods: A Comparison to Methods in Enzymology 6:97–108 (1994)).Concentrations of F(ab)s were determined by quantitative amino acidanalysis. VEGF was coupled to a CM-5 biosensor chip through primaryamine groups according to manufacturer's instructions (Pharmacia).Off-rate kinetics were measured by saturating the chip with F(ab) (35 μlof 2 μM F(ab) at a flow rate of 20 μl/min) and then switching to buffer(PBS-0.05% polysorbate 20). Data points from 0–4500 sec were used foroff-rate kinetic analysis. The dissociation rate constant (_(off)) wasobtained from the slope of the plot of In(RO/R) versus time, where RO isthe signal at t=0 and R is the signal at each time point.

On-rate kinetics were measured using two-fold serial dilutions of F(ab)(0.0625–2 mM). The slope, K_(s), was obtained from the plot ofIn(-dR/dt) versus time for each F(ab) concentration using the BIAcore™kinetics evaluation software as described in the Pharmacia Biosensormanual. R is the signal at time t. Data between 80 and 168, 148, 128,114, 102, and 92 sec were used for 0.0625, 0.125, 0.25, 0.5, 1, and 2 mMF(ab), respectively. The association rate constant (k_(on)) was obtainedfrom the slope of the plot of K_(s) versus F(ab) concentration. At theend of each cycle, bound F(ab) was removed by injecting 5 μl of 50 mMHCl at a flow rate of 20 μl/min to regenerate the chip.

Endothelial Cell Growth Assay: Bovine adrenal cortex-derived capillaryendothelial cells were cultured in the presence of low glucoseDulbecco's modified Eagle's medium (DMEM) (GIBCO) supplemented with 10%calf serum, 2 mM glutamine, and antibiotics (growth medium), essentiallyas previously described (Leung et al. Science 246:1306–1309 (1989)). Formitogenic assays, endothelial cells were seeded at a density of 6×10³cells per well, in 6-well plates in growth medium. Either muMAb VEGFA.4.6.1 or rhuMAb VEGF was then added at concentrations ranging between1 and 5000 ng/ml. After 2–3 hr, purified E. coli-expressed rhVEGF165 wasadded to a final concentration of 3 ng/ml. For specificity control, eachantibody was added to endothelial cells at the concentration of 5000ng/ml, either alone or in the presence of 2 ng/ml bFGF. After five orsix days, cells were dissociated by exposure to trypsin and counted in aCoulter counter (Coulter Electronics, Hialeah, Fla.). The variation fromthe mean did not exceed 10%. Data were analyzed by a four-parametercurve fitting program (KaleidaGraph).

In Vivo Tumor Studies: Human A673 rhabdomyosarcoma cells (ATCC; CRL1598) were cultured as previously described in DMEM/F12 supplementedwith 10% fetal bovine serum, 2 mM glutamine and antibiotics (Kim et al.Nature 362:841–844 (1993) and Borgström et al. Cancer Res. 56:4032–4039(1996)). Female BALB/c nude mice, 6–10 weeks old, were injectedsubcutaneously with 2×10⁶ tumor cells in the dorsal area in a volume of200 μl. Animals were then treated with muMAb VEGF A.4.6.1, rhuMAb VEGFor a control MAb directed against the gp120 protein (Kim et al. Nature362:841–844 (1993)). Both anti-VEGF MAbs were administered at the dosesof 0.5 and 5 mg/kg; the control MAb was given at the dose of 5 mg/kg.Each MAb was administered twice weekly intra peritoneally in a volume of100 μl, starting 24 hr after tumor cell inoculation. Each groupconsisted of 10 mice. Tumor size was determined at weekly intervals.Four weeks after tumor cell inoculation, animals were euthanized and thetumors were removed and weighed. Statistical analysis was performed byANOVA.

Results

Humanization: The consensus sequence for the human heavy chain subgroupIII and the light chain subgroup k I were used as the framework for thehumanization (Kabat et al., supra) (FIGS. 1A and 1B). This framework hasbeen successfully used in the humanization of other murine antibodies(Werther et al., supra; Carter et al., supra; Presta et al. J. Immunol.151:2623–2632 (1993); and Eigenbrot et al. Proteins 18:49–62 (1994)).CDR-H1 included residues H26-H35. The other CDRs were according to Kabatet al., supra. All humanized variants were initially made and screenedfor binding as F(ab)s expressed in E. coli. Typical yields from 500 mlshake flasks were 0.1–0.4 mg F(ab).

The chimeric F(ab) was used as the standard in the binding assays. Inthe initial variant, F(ab)-1, the CDR residues were transferred from themurine antibody to the human framework and, based on the models of themurine and humanized F(ab)s, the residue at position H49 (Ala in human)was changed to the murine Gly. In addition, F(ab)s which consisted ofthe chimeric heavy chain/F(ab)-1 light chain (F(ab)-2) and F(ab)-1 heavychain/chimeric light chain (F(ab)-3) were generated and tested forbinding. F(ab)-1 exhibited a binding affinity greater than 1000-foldreduced from the chimeric F(ab) (Table 2). Comparing the bindingaffinities of F(ab)-2 and F(ab)-3 suggested that framework residues inthe F(ab)-1 VH domain needed to be altered in order to increase binding.

TABLE 2 Binding of Humanized Anti-VEGF F(ab) Variants to VEGF^(a) EC50F(ab)-X EC50 chimeric F(ab)^(c) Variant Template Changes^(b) PurposeMean S.D. N chim- Chimeric 1.0 F(ab) F(ab) F(ab)-1 Human FR Straight CDRswap >1350 2 AlaH49Gly F(ab)-2 Chimera Light Chain >145 3 F(ab)-1 HeavyChain F(ab)-3 F(ab)-1 Light Chain 2.6 0.1 2 Chimera Heavy Chain F(ab)-4F(ab)-1 ArgH71Leu CDR-H2 conformation >295 3 AsnH73Thr Framework F(ab)-5F(ab)-4 LeuL46Val VL-VH interface 80.9 6.5 2 F(ab)-6 F(ab)-5 LeuH78AlaCDR-H1 conformation 36.4 4.2 2 F(ab)-7 F(ab)-5 IleH69Phe CDR-H2conformation 45.2 2.3 2 F(ab)-8 F(ab)-5 IleH69Phe CDR-H2 conformation9.6 0.9 4 LeuH78Ala CDR-H1 conformation F(ab)-9 F(ab)-8 GlyH49Ala CDR-H2conformation >150 2 F(ab)-10 F(ab)-8 AsnH76Ser Framework 6.4 1.2 4F(ab)-11 F(ab)-10 LysH75Ala Framework 3.3 0.4 2 F(ab)-12 F(ab)-10ArgH94Lys CDR-H3 conformation 1.6 0.6 4 ^(a)Anti-VEGF F(ab) variantswere incubated with biotinylated VEGF and then transferred to ELISAplates coated with KDR-IgG (Park et al., supra). ^(b)Murine residues areunderlined; residue numbers are according to Kabat et al., supra.^(c)Mean and standard deviation are the average of the ratios calculatedfor each of the independent assays; the EC50 for chimeric F(ab) was0.049 ± 0.013 mg/ml (1.0 nM).

Changing human residues H71 and H73 to their murine counterparts inF(ab)-4 improved binding by 4-fold (Table 2). Inspection of the modelsof the murine and humanized F(ab)s suggested that residue L46, buried atthe VL-VH interface and interacting with CDR-H3 (FIG. 2), might alsoplay a role either in determining the conformation of CDR-H3 and/oraffecting the relationship of the VL and VH domains. When the murine Valwas exchanged for the human Leu at L46 (F(ab)-5), the binding affinityincreased by almost 4-fold (Table 2). Three other buried frameworkresidues were evaluated based on the molecular models: H49, H69 and H78.Position H69 may affect the conformation of CDR-H2 while position H78may affect the conformation of CDR-H1 (FIG. 2). When each wasindividually changed from the human to murine counterpart, the bindingimproved by 2-fold in each case (F(ab)-6 and F(ab)-7, Table 2). Whenboth were simultaneously changed, the improvement in binding was 8-fold(F(ab)-8, Table 2). Residue H49 was originally included as the murineGly; when changed to the human consensus counterpart Ala the binding wasreduced by 15-fold (F(ab)-9, Table 2).

In F(ab)-10 and F(ab)-11 two residues in framework loop 3, FR-3, werechanged to their murine counterparts: AsnH76 to murine Ser (F(ab)-10)and LysH75 to murine Ala (F(ab)-11). Both effected a relatively smallimprovement in binding (Table 2). Finally, at position H94 human andmurine sequences most often have an Arg (Kabat et al., supra). InF(ab)-12, this Arg was replaced by the rare Lys found in the murineantibody (FIG. 1A) and this resulted in binding which was less than2-fold from the chimeric F(ab) (Table 2). F(ab)-12 was also compared tothe chimeric F(ab) using the BIAcore™ system (Pharmacia). Using thistechnique the K_(d) of the humanized F(ab)-12 was 2-fold weaker thanthat of the chimeric F(ab) due to both a slower k_(on) and fasterk_(off) (Table 3).

TABLE 3 Binding of Anti-VEGF F(ab) Variants to VEGF Using the BIAcore ™System^(a) Amount of (Fab) bound k_(off) k_(on) K_(d) Variant (RU) (s⁻¹⁾(M-1s-1) (nM) chim- 4250 5.9 × 10-5 6.5 × 104 0.91 F(ab)b F(ab)-12 37406.3 × 10-5 3.5 × 104 1.8 ^(a)The amount of F(ab) bound, in resonanceunits (RU), was measured using a BIAcore ™ system when 2 μg F(ab) wasinjected onto a chip containing 2480 RU immobilized VEGF. Off-ratekinetics (k_(off)) were measured by saturating the chip with F(ab) andthen monitoring dissociation after switching to buffer. On-rate kinetics(k_(on)) were measured using two-fold serial dilutions of F(ab). K_(d),the equilibrium dissociation constant, was calculated as k_(off)/k_(on).^(b)chim-F(ab) is a chimeric F(ab) with murine VL and VH domains fusedto human CL and CH1 heavy domains.

Full length mAbs were constructed by fusing the VL and VH domains of thechimeric F(ab) and variant F(ab)-12 to the constant domains of human klight chain and human IgG1 heavy chain. The full length 12-IgG1(F(ab)-12 fused to human IgG1) exhibited binding which was 1.7-foldweaker than the chimeric IgG1 (Table 4). Both 12-IgG1 and the chimericIgG1 bound slightly less well than the original murine mAb A4.6.1 (Table4).

TABLE 4 Binding of Anti-VEGF IgG Variants to VEGF^(a) IgG1/chIgG1^(b)Variant Mean S.D. N chIgG1 1.0 2 murIgG1c 0.759 0.001 2 12-IgG1d 1.710.03 2 ^(a)Anti-VEGF IgG variants were incubated with biotinylated VEGFand then transferred to ELISA plates coated with KDR-IgG (Park et al.,(1994), supra). ^(b)chIgG1 is chimeric IgG1 with murine VL and VHdomains fused to human CL and IgG1 heavy chains; the EC50 for chIgG1 was0.113 ± 0.013 μg/ml (0.75 nM). ^(c)murIgG1 is muMAbVEGF A461 purifiedfrom ascites. ^(d)12-IgG1 is F(ab)-12 VL and VH domains fused to humanCL and IgG1 heavy chains.

Biological Studies: rhuMAb VEGF and muMAb VEGF A.4.6.1. were comparedfor their ability to inhibit bovine capillary endothelial cellproliferation in response to a near maximally effective concentration ofVEGF (3 ng/ml). As illustrated in FIG. 3, the two MAbs were essentiallyequivalent, both in potency and efficacy. The ED50 values wererespectively 50±5 ng/ml and 48±8 ng/ml (˜0.3 nM). In both cases 90%inhibition was achieved at the concentration of 500 ng/ml (˜3 nM).Neither muMAb VEGF A.4.6.1 nor rhuMAb VEGF had any effect on basal orbFGF-stimulated proliferation of capillary endothelial cells, confirmingthat the inhibition is specific for VEGF.

To determine whether such equivalency applies also to an in vivo system,the two antibodies were compared for their ability to suppress thegrowth of human A673 rhabdomyosarcoma cells in nude mice. Previousstudies have shown that muMAb VEGF A.4.6.1 has a dramatic inhibitoryeffect in this tumor model (Kim et al. Nature 362:841–844 (1993) andBorgström et al. Cancer Res 56:4032–4039 (1996)). As shown in FIG. 4, atboth doses tested (0.5 and 5 mg/kg), the two antibodies markedlysuppressed tumor growth as assessed by tumor weight measurements fourweeks after cell inoculation. The decreases in tumor weight compared tothe control group were respectively 85% and 93% at each dose in theanimals treated with muMAb VEGF A.4.6.1. versus 90% and 95% in thosetreated with rhuMAb VEGF. Similar results were obtained with the breastcarcinoma cell line MDA-MB 435.

EXAMPLE 2

In this example, the murine anti-VEGF antibody A4.6.1 discussed abovewas humanized by randomizing a small set of framework residues and bymonovalent display of the resultant library of antibody molecules on thesurface of filamentous phage in order to identify high affinityframework sequences via affinity-based selection.

Materials and Methods

Construction of Anti-VEGF Phagemid Vector, pMB4–19: The murine anti-VEGFmAb A4.6.1 is discussed above in Example 1. The first Fab variant ofhumanized A4.6.1, hu2.0, was constructed by site-directed mutagenesisusing a deoxyuridine-containing template of plasmid pAK2 (Carter et al.Proc. Natl. Acad. Sci. U.S.A. 89:4285–4289 (1992)) which codes for ahuman V_(L)κI-Cκ₁ light chain and human V_(H)III-C_(H)1 γ₁ heavy chainFd fragment The transplanted A4.6.1 CDR sequences were chosen accordingto the sequence definition of Kabat et al., supra, except for CDR-H1which included residues 26–35. The Fab encoding sequence was subclonedinto the phagemid vector phGHamg3 (Bass et al. Proteins 8:309–314 (1990)and Lowman et al. Biochemistry 30:10832–10838 (1991)). This construct,pMB4-19, encodes the initial humanized A4.6.1 Fab, hu2.0, with theC-terminus of the heavy chain fused precisely to the carboxyl portion ofthe M13 gene III coat protein. pMB4-19 is similar in construction topDH188, a previously described plasmid for monovalent display of Fabfragments (Garrard et al. Biotechnology 9:1373–1377 (1991)). Notabledifferences between pMB4-19 and pDH188 include a shorter M13 gene IIIsegment (codons 249–406) and use of an amber stop codon immediatelyfollowing the antibody heavy chain Fd fragment. This permits expressionof both secreted heavy chain or heavy chain-gene III fusions in supEsuppressor strains of E. coli.

Expression and Purification of Humanized A4.6.1 Fab Fragment: E. colistrain 34B8, a nonsuppressor, was transformed with phagemid pMB4–19, orvariants thereof. Single colonies were grown overnight at 37° C. in 5 mL2YT containing 50 μg/mL carbenicillin. These cultures were diluted into200 mL AP5 medium (Chang et al. Gene 55:189–196 (1987)) containing 20μg/mL carbenicillin and incubated for 26 hr at 30° C. The cells werepelleted at 4000×g and frozen at −20° C. for at least 2 h. Cell pelletswere then resuspended in 5 mL of 10 mM Tris-HCl (pH 7.6) containing 1 mMEDTA, shaken at 4° C. for 90 min and centrifuged at 10,000×g for 15 min.The supernatant was applied to a 1 mL streptococcal protein G-sepharosecolumn (Pharmacia) and washed with 10 mL of 10 mM MES (pH 5.5). Thebound Fab fragment was eluted with 2.5 mL 100 mM acetic acid andimmediately neutralized with 0.75 mL 1M Tris-HCl, pH 8.0. Fabpreparations were buffer-exchanged into PBS and concentrated usingCentricon-30 concentrators (Amicon). Typical yields of Fab were ˜1 mg/Lculture, post-protein G purification. Purified Fab samples werecharacterized by electrospray mass spectrometry, and concentrations weredetermined by amino acid analysis.

Construction of the Anti-VEGF Fab Phagemid Library: The humanized A4.6.1phagemid library was constructed by site-directed mutagenesis accordingto the method of Kunkel et al. Methods Enzymol. 204:125–139 (1991)). Aderivative of pMB4-19 containing TAA stop triplets at V_(H) codons 24,37, 67 and 93 was prepared for use as the mutagenesis template (allsequence numbering according to Kabat et al., supra). This modificationwas to prevent subsequent background contamination by wild typesequences. The codons targeted for randomization were 4 and 71 (lightchain) and 24, 37, 67, 69, 71, 73, 75, 76, 78, 93 and 94 (heavy chain).

In order to randomize heavy chain codons 67, 69, 71, 73, 75, 76, 78, 93and 94 with a single mutagenic oligonucleotide, two 126-meroligonucleotides were first preassembled from 60 and 66-mer fragments bytemplate-assisted enzymatic ligation. Specifically, 1.5 nmol of 5′phosphorylated oligonucleotide 503-1 (5′-GAT TTC AAA CGT CGT NYT ACT WTTTCT AGA GAC AAC TCC AAA AAC ACA BYT TAC CTG CAG ATG AAC-3′ (SEQ IDNO:22)) or 503-2 (5′-GAT TTC AAA CGT CGT NYT ACT WTT TCT TTA GAC ACC TCCGCA AGC ACA BYT TAC CTG CAG ATG AAC-3′ (SEQ ID NO:23)) were combinedwith 1.5 nmol of 503-3 (5′-AGC CTG CGC GCT GAG GAC ACT GCC GTC TAT TACTGT DYA ARG TAC CCC CAC TAT TAT GGG-3′ (SEQ ID NO:24)) (randomizedcodons underlined; N=A/G/T/C; W=A/T; B=G/T/C; D=G/A/T; R=A/G; Y=C/T).Then, 1.5 nmol of template oligonucleotide (5′-CTC AGC GCG CAG GCT GTTCAT CTG CAG GTA-3′ (SEQ ID NO:25)), with complementary sequence to the5′ ends of 503-1/2 and the 3′ end of 503–3, was added to hybridize toeach end of the ligation junction. Taq ligase (thermostable ligase fromNew England Biolabs) and buffer were added, and the reaction mixture wassubjected to 40 rounds of thermal cycling, (95° C. 1.25 min; 50° C. for5 min) so as to cycle the template oligonucleotide between ligated andunligated junctions. The product 126-mer oligonucleotides were purifiedon a 6% urea/TBE polyacrylamide gel and extracted from thepolyacrylamide in buffer. The two 126-mer products were combined inequal ratio, ethanol precipitated and finally solubilized in 10 mMTris-HCl, 1 mM EDTA. The mixed 126-mer oligonucleotide product waslabeled 504-01.

Randomization of select framework codons (V_(L) 4, 71; V_(H) 24, 37, 67,69, 71, 73, 75, 76, 93, 94) was effected in two steps. Firstly, V_(L)randomization was achieved by preparing three additional derivatives ofthe modified pMB4–19 template. Framework codons 4 and 71 in the lightchain were replaced individually or pairwise using the two mutagenicoligonucleotides 5′-GCT GAT ATC CAG TTG ACC CAG TCC CCG-3′ (SEQ IDNO:26) 5′-and TCT GGG ACG GAT TAC ACT CTG ACC ATC-3′ (SEQ ID NO:27).Deoxyuridine-containing template was prepared from each of these newderivatives. Together with the original template, these four constructscoded for each of the four possible light chain framework sequencecombinations (Table 5).

Oligonucleotides 504-1, a mixture of two 126-mer oligonucleotides (seeabove), and 5′-CGT TTG TCC TGT GCA RYT TCT GGC TAT ACC TTC ACC AAC TATGGT ATG AAC TGG RTC CGT CAG GCC CCG GGT AAG-3′ (SEQ ID NO:28) were usedto randomize heavy chain framework codons using each of the fourtemplates just described. The four libraries were electroporated into E.coli XL-1 Blue cells (Stratagene) and combined. The total number ofindependent transformants was estimated at >1.2×10⁸, approximately1,500-fold greater than the maximum number of DNA sequences in thelibrary.

A variety of systems have been developed for the functional display ofantibody fragments on the surface of filamentous phage. Winter et al.,Ann. Rev. Immunol. 12,433 (1994). These include the display of Fab orsingle chain Fv (scFv) fragments as fusions to either the gene III orgene VIII coat proteins of M13 bacteriophage. The system selected hereinis similar to that described by Garrard et al., Biotechn, 9, 1373 (1991)in which a Fab fragment is monovalently displayed as a gene III fusion(FIG. 7). This system has two notable features. In particular, unlikescFvs, Fab fragments have no tendency to form dimeric species, thepresence of which can prevent selection of the tightest binders due toavidity effects. Additionally the monovalency of the displayed proteineliminates a second potential source of avidity effects that wouldotherwise result from the presence of multiple copies of a protein oneach phagemid particle. Bass and Wells, Proteins 8:309 (1990) and Lowmanet al., Biochemistry 30:10832 (1991).

Phagemid particles displaying the humanized A4.6.1 Fab fragments werepropagated in E. coli XL-1 Blue cells. Briefly, cells harboring therandomized pMB4-19 construct were grown overnight at 37° C. in 25 mL 2YTmedium containing 50 μg/mL carbenicillin and approximately 10¹⁰ M13KO7helper phage (Vieira & Messing Methods Enzymol. 153:3–11 (1987)).Phagemid stocks were purified from culture supernatants by precipitationwith a saline polyethylene glycol solution, and resuspended in 100 μLPBS (˜10¹⁴ phagemid/mL)

Selection of Humanized A4.6. 1 Fab Variants: Purified VEGF₁₂₁ (100 μL at10 μg/mL in PBS) was coated onto a microtiter plate well overnight at 4°C. The coating solution was discarded and this well, in addition to anuncoated well, were blocked with 6% skim milk for 1 h and washed withPBS containing 0.05% TWEEN 20™ (detergent). Then, 10 μL of phagemidstock, diluted to 100 μL with 20 mM Tris (pH 7.5) containing 0.1% BSAand 0.05% TWEEN 20™, was added to each well. After 2 hours the wellswere washed and the bound phage eluted with 100 μL of 0.1 M glycine (pH2.0), and neutralized with 25 μL of 1M Tris pH 8.0. An aliquot of thiswas used to titer the number of phage eluted. The remaining phage elutedfrom the VEGF-coated well were propagated for use in the next selectioncycle. A total of 8 rounds of selection was performed after which time20 individual clones were selected and sequenced (Sanger et al. Proc.Natl. Acad. Sci. U.S.A. 74:5463–5467 (1977)).

Determination of VEGF Binding Affinities: Association (k_(on)) anddissociation (k_(off)) rate constants for binding of humanized A4.6.1Fab variants to VEGF₁₂₁ were measured by surface plasmon resonance(Karlsson et al. J. Immun. Methods 145:229–240 (1991)) on a PharmaciaBIAcore instrument. VEGF₁₂₁ was covalently immobilized on the biosensorchip via primary amino groups. Binding of humanized A4.6.1 Fab variantswas measured by flowing solutions of Fab in PBS/0.05% TWEEN 20™ over thechip at a flow rate of 20 μL/min. Following each binding measurement,residual Fab was stripped from the immobilized ligand by washing with 5μL of 50 mM aqueous HCl at 3 μL/min. Binding profiles were analyzed bynonlinear regression using a simple monovalent binding model(BIAevaluation software v2.0; Pharmacia).

Results

Construction of Humanized A4.6.1: An initial humanized A4.6.1 Fabfragment was constructed (hu2.0, FIGS. 5A and 5B), in which the CDRsfrom A4.6.1 were grafted onto a human V_(L)κI-V_(H)III framework. Allother residues in hu2.0 were maintained as the human sequence. Bindingof this variant to VEGF was so weak as to be undetectable. Based on therelative affinity of other weakly-binding humanized A4.6.1 variants, theK_(D) for binding of hu2.0 was estimated at >7 μM. This contrasts withan affinity of 1.6 nM for a chimeric Fab construct consisting of theintact V_(L) and V_(H) domains from murine A4.6.1 and human constantdomains. Thus binding of hu2.0 to VEGF was at least 4000-fold reducedrelative to the chimera.

Design of Antibody Library: The group of framework changes to the humanframework sequence herein is shown in Table 5 and FIG. 6.

TABLE 5 Key Framework Residues Important for Antigen Binding andTargeted for Randomization Human Vκ_(L)I, V_(H)III consensus MurineA4.6.1 Framework residue residue residue Randomizationa V_(L): 4 Met MetMet, Leu 71 Phe Tyr Phe, Tyr V_(H): 24 Ala Ala Ala, Val, Thr 37 Val ValVal, Ile 67 Phe Phe Phe, Val, Thr, Leu, Ile, Ala 69 Ile Phe Ile, Phe 71Arg Leu Arg^(b), Leu^(b) 73 Asp Thr Asp^(b), Thr^(b) 75 Lys Ala Lys^(b),Ala^(b) 76 Asn Ser Asn^(b), Ser^(b) 78 Leu Ala Leu, Ala, Val, Phe 93 AlaAla Ala, Val, Leu, Ser, Thr 94 Arg Lys Arg, Lys ^(a)Amino acid diversityin phagemid library ^(b)V_(H)71, 73, 75, 76 randomized to yield theall-murine (L71/T73/A75/S76) or all-human (R71/D73/K75/N76) V_(H)IIItetrad

A concern in designing the humanized A4.6.1 phagemid library was thatresidues targeted for randomization were widely distributed across theV_(L) and V_(H) sequences. Limitations in the length of syntheticoligonucleotides requires that simultaneous randomization of each ofthese framework positions can only be achieved through the use ofmultiple oligonucleotides. However, as the total number ofoligonucleotides increases, the efficiency of mutagenesis decreases(i.e. the proportion of mutants obtained which incorporate sequencederived from all of the mutagenic oligonucleotides). To circumvent thisproblem, two featires were incorporated into the library construction.The first was to prepare four different mutagenises templates coding foreach of the possible V_(L) framework combinations. This was simple to dogiven the limited diversity of the light chain framework (only 4different sequences), but was beneficial in that it eliminated the needfor two oligonucleotides from the mutagenesis strategy. Secondly, two126-base oligonucleotides were preassembled from smaller syntheticfragments. This made possible randomization of V_(H) codons 67, 69, 71,73, 75, 76, 93 and 94 with a single long oligonucleotide, rather thantwo smaller ones. The final randomization mutagenesis strategy thereforeemployed only two oligonucleotides simultaneously onto four differenttemplates.

Selection of Tight Binding Humanized A4.6. 1 Fab's: Variants from thehumanized A4.6.1 Fab phagemid library were selected based on binding toVEGF. Enrichment of functional phagemid, as measured by comparing titersfor phage eluted from a VEGF-coated versus uncoated microtiter platewell, increased up to the seventh round of affinity panning. After oneadditional round of sorting, 20 clones were sequenced to identifypreferred framework residues selected at each position randomized. Theseresults, summarized in Table 6, revealed strong consensus amongst theclones selected. Ten out of the twenty clones had the identical DNAsequence, designated hu2.10. Of the thirteen framework positionsrandomized, eight substitutions were selected in hu2.10 (V_(L) 71; V_(H)37, 71, 73, 75, 76, 78 and 94). Interestingly, residues V_(H) 37 (IIe)and 78 (Val) were selected neither as the human V_(H)III or murineA4.6.1 sequence. This result suggests that some framework positions maybenefit from extending the diversity beyond the target human and parentmurine framework sequences.

TABLE 6 Sequences Selected from the Humanized A4.6.1 Phagemid FabLibrary Residue substitutions V_(L) V_(H) Variant 4 71 24 37 67 69 71 7375 76 78 93 94 murine M Y A V F F L T A S A A K A4.6.1 hu2.0 M F A V F IR N K N L A R (CDR- graft) Phage-selected clones: hu2.1(2) — Y — I — — —— — — V — K hu2.2(2) L Y — I — — — — — — V — K hu2.6(1) L — — I T — L TA S V — K hu2.7(1) L — — I — — — — — — V — K hu2.10(10) — Y — I — — L TA S V — K Differences between hu2.0 and murine A4.6.1 antibodies areunderlined. The number of identical clones identifies for eachphage-selected sequence is indicated in parentheses. Dashes in thesequences of phage-selected clones indicate selection of the humanV_(L)κI-V_(H)III framework sequence (i.e. as in hu2.0).

There were four other unique amino acid sequences among the remainingten clones analyzed: hu2.1, hu2.2, hu2.6 and hu2.7. All of these clones,in addition to hu2.10, contained identical framework substitutions atpositions V_(H) 37 (IIe), 78 (Val) and 94 (Lys), but retained the humanV_(H)III consensus sequence at positions 24 and 93. Four clones had lostthe light chain coding sequence and did not bind VEGF when tested in aphage ELISA assay (Cunningham et al. EMBO J. 13:2508–251 (1994)). Suchartifacts can often be minimized by reducing the number of sortingcycles or by propagating libraries on solid media.

Expression and Binding Affinity of Humanized A4.6.1 Variants:Phage-selected variants hu2.1, hu2.2, hu2.6, hu2.7 and hu2.10 wereexpressed in E. coli using shake flasks and Fab fragments were purifiedfrom periplasmic extracts by protein G affinity chromatography.Recovered yields of Fab for these five clones ranged from 0.2 (hu2.6) to1.7 mg/L (hu2.1). The affinity of each of these variants for antigen(VEGF) was measured by surface plasmon resonance on a BIAcore instrument(Table 7). Analysis of this binding data revealed that the consensusclone hu2.10 possessed the highest affinity for VEGF out of the fivevariants tested. Thus the Fab phagemid library was selectively enrichedfor the tightest binding clone. The calculated K_(D) for hu2.10 was 55nM, at least 125-fold tighter than for hu2.0 which contains no frameworkchanges (K_(D)>7 μM). The other four selected variants all exhibitedweaker binding to VEGF, ranging down to a K_(D) of 360 nM for theweakest (hu2.7). Interestingly, the K_(D) for hu2.6, 67 nM, was onlymarginally weaker than that of hu2.10 and yet only one copy of thisclone was found among 20 clones sequenced. This may have due to a lowerlevel of expression and display, as was the case when expressing thesoluble Fab of this variant. However, despite the lower expression rate,this variant is useful as a humanized antibody.

TABLE 7 VEGF Binding Affinity of Humanized A4.6.1 Fab Variants$\begin{matrix}k_{on} \\{{M^{- 1}s} -^{1}{\text{/}10^{4}}}\end{matrix}\quad$ $\begin{matrix}k_{off} \\{10^{4}s^{- 1}}\end{matrix}\quad$ $\begin{matrix}K_{D} \\{nM}\end{matrix}\quad$$\frac{K_{D}\left( {{A4}{.6}{.1}} \right)}{K_{D}({mut})}$ Variant A4.6.1chimera 5.4  0.85 1.6 >4000 hu2.0 ND ND >7000** Phage selected clones:hu2.1 0.70 18 260 170 hu2.2 0.47 16 340 210 hu2.6 0.67 4.5 67 40 hu2.70.67 24 360 230 hu2.10 0.63 3.5 55 35 *hu2.10V 2.0  1.8 9.3 5.8 *hu2.10V= hu2.10 with mutation V_(L)Leu -> Val Estimated errors in the Biacorebinding measurements are +/−25%. **Too weak to measure; estimate oflower bound

Additional Improvement of Humanized Variant hu2.1: Despite the largeimprovement in antigen affinity over the initial humanized variant,binding of hu2.10 to VEGF was still 35-fold weaker than a chimeric Fabfragment containing the murine A4.6.1 V_(L) and V_(H) domains. Thisconsiderable difference suggested that further optimization of thehumanized framework might be possible through additional mutations. Ofthe Vernier residues identified by Foote & Winter J. Mol. Biol.224:487–499 (1992), only residues V_(L) 46, V_(H) 2 and V_(H) 48differed in the A4.6.1 versus human V_(L)κI-V_(H)III framework (FIGS. 5Aand 5B) but were not randomized in our phagemid library. A molecularmodel of the humanized A4.6.1 Fv fragment showed that V_(L) 46 sits atthe V_(L)–V_(H) interface and could influence the conformation ofCDR-H3. Furthermore, this amino acid is almost always leucine in mostV_(L)κ frameworks (Kabat et al., supra), but is valine in A4.6.1.Accordingly, a Leu -> Val substitution was made at this position in thebackground of hu2.10. Analysis of binding kinetics for this new variant,hu2.10V, indicated a further 6-fold improvement in the K_(D) for VEGFbinding, demonstrating the importance of valine at position V_(L) 46 inantibody A4.6.1. The K_(D) for hu2.10V (9.3 nM) was thus within 6-foldthat of the chimera. In contrast to V_(L) 46, no improvement in thebinding affinity of hu2.10 was observed for replacement of either V_(H)2 or V_(H) 48 with the corresponding residue from murine A4.6.1.

EXAMPLE 3

In this example, CDR randomization, affinity maturation by monovalentFab phage display, and cumulative combination of mutations were used toenhance the affinity of a humanized anti-VEGF antibody.

Construction of Humanized Antibody pY0101: Phage-displayed antibodyvector phMB4-19-1.6 (see FIGS. 8A–E) was used as a parent. In thisconstruct, anti-VEGF is expressed as a Fab fragment with its heavy chainfused to the N-terminus of the truncated g3p. Both the light and heavychains are under the control of phoA promoter with an upstream stIIsignal-sequence for secretion into the periplasm. Point mutationsoutside the CDR regions were made by site-directed mutagenesis toimprove affinity for VEGF with oligonucleotides HL-242, HL-243, HL-245,HL-246, HL-254, HL-256, and HL-257 as shown in Table 8 below:

TABLE 8 Oligos for Directed Mutations Oligo Substitution/ Number RegionComments Sequence HL-242 VL M4L 5′-GATATCCAGTTGACCCAGTCCCCG-3′ (SEQ IDNO:29) HL-243 VL L46V 5′-GCTCCGAAAGTACTGATTTAC-3′ (SEQ ID NO:30) HL-245VH CDR-7 5′-CGTCGTTTCACTTTTTCTGCAGACACCT (SEQ ID NO:31)CCAGCAACACAGTATACCTGCAGATG-3′ HL-246 VH R98K5′-CTATTACTGTGCAAAGTACCCCCAC-3′ (SEQ ID NO:32) HL-254 VL Y71F5′-GGGACGGATTTCACTCTGACCATC-3′ (SEQ ID NO:33) HL-256 VH I37V5′-GGTATGAACTGGGTCCGTCAGGCCCC- (SEQ ID NO:34) 3′ HL-257 VH CDR-75′-CGTCGTTTCACTTTTTCTTTAGACACCT (SEQ ID NO:35) A72LCCAAAAGCACAGCATACCTGCAGATGAAC- S76K 3′ N77S

The resulting variant was termed Y0101 (FIGS. 9A and 9B).

Construction of the First Generation of Antibody-Phage Libraries: Toprevent contamination by wild-type sequence, templates with the TAA stopcodon at the targeted sites for randomization were prepared and used forconstructing libraries by site-directed mutagenesis witholigonucleotides using the degenerate NNS codon (where N is an equalmixture of A, G, C, and T while S is an equal mixture of G and C) forsaturation mutagenesis. VL1 and VH3 were chosen as potential candidatesfor affinity enhancement (FIGS. 9A and B) . Within the CDRs, twolibraries were constructed from the pY0101 template. VL1 was mutatedusing stop-template oligonucleotides HL-248 and HL-249 (Table 9) andlibrary oligonucleotides HL-258 and HL-259 (Table 10). Similarly, threelibraries were constructed for VH3 using stop template oligonucleotidesHL-250, HL-251, and HL-252 (Table 9), and library oligonucleotidesHL-260, HL-261, and HL-262 (Table 10). Library construction issummarized in Tables 9 and 10 below.

TABLE 9 Template Oligos for Mutagenesis Oligo Region Number CommentsSequence HL-248 VL1 5′-GGGTCACCATCACCTGCTAAGCATAATAATAATAAAGCAACTA (SEQID NO:36) TTTAAACTGG-3′ HL-249 VL15′-GCGCAAGTCAGGATATTTAATAATAATAATAATGGTATCAACAG (SEQ ID NO:37)AAACCAGG-3′ HL-250 VH3 5′-GTCTATTACTGTGCAAAGTAATAACACTAATAAGGGAGCAGCC(SEQ ID NO:38) ACTGG-3′ HL-251 VH35′-GGTACCCCCACTATTATTAATAATAATAATGGTATTTCGACGTC (SEQ ID NO:39) TGGGG-3′HL-252 VH3 5′-CACTATTATGGGAGCAGCCACTAATAATAATAAGTCTGGGTCA (SEQ ID NO:40)AGGAACCCTG-3′ HL-263 VH1 5′-TCCTGTGCAGCTTCTGGCTAATAATTCTAATAATAAGGTATGAA(SEQ ID NO:41) CTGGGTCCG-3′ HL-264 VH25′-GAATGGGTTGGATGGATTAACTAATAATAAGGTTAACCGACCT (SEQ ID NO:42)ATGCTGCGG-3′ YC-80 VH3 5′-CTGTGCAAAGTACCCGTAATATTAATAATAATAACACTGGTATT(SEQ ID NO:43) TCGAC-3′ YC-100 CDR75′-CGTTTCACTTTTTCTTAAGACTAATCCAAATAAACAGCATACCT (SEQ ID NO:44) GCAG-3′YC-102 VH2 5′-GAATGGGTTG GATGGATTTAATAATAATAAGGTGAACCGACCT (SEQ IDNO:45) ATG-3′

TABLE 10 Random Oligos for Library Construction Oligo Region NumberComment Sequence HL-258 VL1 5′-GGGTCACCATCACCTGCNNSGCANNSNNSNNSNNSAGC(SEQ ID NO:46) AACTATTTAAACTGG-3′ HL-259 VL15′-GCGCAAGTCAGGATATTNNSNNSNNSNNSNNSTGGTATCAACA (SEQ ID NO:47)GAAACCAGG-3′ HL-260 VH3 5′-GTCTATTACTGTGCAAAGNNSNNSCACNNSNNSGGGAGCAGC(SEQ ID NO:48) CACTGG-3′ HL-261 VH35′-GGTACCCCCACTATTATNNSNNSNNSNNSTGGTATTTCGACGT (SEQ ID NO:49) CTGGGG-3′HL-262 VH3 5′-CACTATTATGGGAGCAGCCACNNSNNSNNSNNSGTCTGGGGT (SEQ ID NO:50)CAAGGAACCCTG-3′ HL-265 VH15′-TCCTGTGCAGCTTCTGGCNNSNNSTTCNNSNNSNNSGGTATGA (SEQ ID NO:51)ACTGGGTCCG-3′ HL-266 VH2 5′-GAATGGGTTGGATGGATTAACNNSNNSNNSGGTNNSCCGACC(SEQ ID NO:52) TATGCTGCGG-3′ YC-81 VH35′-CTGTGCAAAGTACCCGNNSTATNNSNNSNNSNNSCACTGGTAT (SEQ ID NO:53) TTCGAC-3′YC-101 CDR7 5′-CGTTTCACTTTTTCTNNSGACNNSTCCAAANNSACAGCATACCT (SEQ IDNO:54) GCAG-3′ YC-103 VH2 5′-GAATGGGTTGGATGGATTNNSNNSNNSNNSGGTGAACCGACC(SEQ ID NO:55) TATG-3′

The products of random mutagenesis reactions were electroporated intoXL1-Blue E. coli cells (Stratagene) and amplified by growing 15–16 hwith M13KO7 helper phage. The complexity of each library, ranging from2×10⁷ to 1.5×10⁸, was estimated based upon plating of the initialtransformation onto carbenicillin plates.

Initial Affinity Selections: For each round of selection, approximately10⁹–10¹⁰ phage were screened for binding to plates (Nunc Maxisorp96-well) coated with 2 μg/mL VEGF (recombinant; residue 9-109 version)in 50 mM carbonate buffer, pH 9.6 and blocked with 5% instant milk in 50mM carbonate buffer, pH 9.6. After 1–2 hour binding at room temperature,in the presence of 0.5% bovine serum albumin and 0.05% TWEEN 20™ in PBS,the phage solution was removed, and the plate was washed ten times withPBS/TWEEN™ (0.05% TWEEN 20™ in PBS buffer). Typically, to select forenhanced affinity variants with slower dissociation rates, the plateswere incubated with PBS/TWEEN™ buffer for a period of time whichlengthened progressively for each round of selection (from 0 minute forthe first round, to 3 h for the ninth round of selection). After thePBS/TWEEN™ buffer was removed, the remained phages were eluted with 0.1M HCl and immediately neutralized with 1/3 volume of 1 M Tris, pH 8.0.The eluted phages were propagated by infecting XL1-Blue E. coli cells(Stratagene) for the next selection cycle.

Sequencing data revealed that both VL1 libraries, even after theeighth/ninth round of sorting, remained diverse, tolerating various typeof residues at the sites of randomization. In contrast, the VH3libraries retained only wild type residues or had very conservativesubstitutions. This suggested that the VL1 was more exposed to solventand lay outside the binding interface. In contrast, VH3 did not showdramatically different sidechain substitutions, and therefore might bemore intimately involved in antigen binding.

Phage-ELISA Assay of Binding Affinities: From each of these libraries,representative clones (those represented by abundant sequences) wereassayed for their affinities relative to that of parent clone pY0101 ina phage-ELISA assay. In such an assay, phages were first seriallydiluted to determine a fractional saturation titer which was then heldconstant and used to incubate with varying concentrations of VEGF(starting at 200 nM to 0 nM) in solution. The mixture was thentransferred onto plate precoated with VEGF (2 μg/mL) and blocked with 5%instant milk, and allowed to equilibrate for 1 hour at room temperature.Thereafter, the phage solution was removed and the remaining boundphages were detected with a solution of rabbit anti-phage antibody mixedwith goat anti-rabbit conjugate of horse radish peroxidase. After anhour incubation at room temperature, the plate was developed with achromogenic substrate, o-phenylenediamine (Sigma). The reaction wasstopped with addition of ½ volume of 2.5 M H₂SO₄. Optical density at 492nm was measured on a spectrophotometric plate reader.

Although all of the selected clones from these five libraries showedeither weaker or similar affinities than that of wild type pY0101 inphage-ELISA assay, one particular variant (pY0192) from library HL-258displayed an apparent advantage (about 10 fold) in the level ofexpression or phage display relative to pY0101. This clone containedmutations S24R, S26N, Q27E, D28Q, and I29L in the VL region (FIG. 9A).In addition, this variant was found to have a spurious mutation, M34I,in VH. This variant showed no significant difference in binding affinityto VEGF as compared with the pY0101 variant. To improve the level ofFab-display on phage, and the signal-to-noise ratio for phage-ELISAassays, the corresponding substitutions in pY0192 at VL1 wereincorporated into the template background for constructing both CDRAla-mutants and the second generation of anti-VEGF libraries.

Ala-Scanning the CDRs of Anti-VEGF: To determine the energeticscontributed by each of the amino acids in the CDR regions and thusbetter select target residues for randomization, the CDR regions werescreened by substituting alanine for each residue. Each Ala mutant wasconstructed using site-directed mutagenesis with a syntheticoligonucleotide encoding for the specific alanine substitution. WhereAla was the wild-type residue, Ser was substituted to test the effect ofa sidechain substitution. Phage clones having a single Ala mutation werepurified and assayed in phage-ELISA as described above. Results of theAla-scan demonstrated that Ala-substitution at various positions canhave an effect, ranging from 2 to >150 fold reductions, on antigenbinding affinity compared to pY0192. In addition, it confirmed aprevious observation that VH3, but not VL1, was involved in antigenbinding.

Results of the CDR Ala-scan are summarized in Table 11 below.

TABLE 11 Relative VEGF Affinities of Ala-Scan Fab Variants Residue IC50(mut) Residue IC50 (mut) VL IC50 (wt) VH IC50 (wt) R24A 1 G26A 2 A25S 1Y27A 34 N26A 1 T28A 1 E27A 1 F29A 16 Q28A 1 T30A 1 L29A 1 N31A >150 S30A2 Y32A >150 N31A 2 G33A 6 Y32A 2 I34A 6 L33A 2 N35A 66 N34A 4 W50A >150F50A 1 I51A 4 T51A 1 N52A >150 S52A 1 T53A 9 S53A 1 Y54A 9 L54A 1 T55A 4H55A 1 G56A 1 S56A 1 E57A 2 P58A 1 Q89A 4 T59A 3 Q90A 3 Y60A 2 Y91A 14A61S 1 S92A 1 A62S 1 T93A 1 D63A 1 V94A 2 F64A 1 P95A 3 K65A 1 W96A >150R66A 1 T97A 1 Y99A >150 P100A 38 H101A 4 Y102A 4 Y103A 5 G104A 2 S105A 1S106A >150 H107A 2 W108A >150 Y109A 19 F110A 25 D111A 2All variants are in the background of pY0192 (“wt”; see FIGS. 9A–B).IC50's were determined in a competitive phage-ELISA assay.

The largest effects of Ala substitutions are seen in CDRs H1, H2, andH3, including Y27A (34-fold reduction in affinity), N31A, Y32A, W50A,N52A, Y99A, S106A and W108A (each >150-fold reduction); N35A (66-foldreduction), P100A (38-fold reduction) and F110A (25-fold reduction). Incontrast, only one VL substitution had a large impact on bindingaffinity, W96A (>150-fold reduction). These results point to the threeVH CDRs as the main energetic determinants of Fab binding to VEGF, withsome contribution from VL3.

Design of Second-Generation CDR Mutation Libraries: Two additionallibraries which randomized existing residues in anti-VEGF version Y0192were designed based upon inspection of the crystal structure. In VH2,residues 52–55 were randomized because they lie within the bindinginterface with VEGF. An additional region of the Fab, termed “CDR7” (seeFIG. 10B), was also targeted for randomization because several residuesin this loop, while not contacting VEGF, do have contacts with the VHloops of the antibody. These represented potential sites for affinityimprovement through secondary effects upon the interface residues.Residues L72, T74, and S77 were randomized in this CDR7 library.

Also based upon the crystal structure, one of the original CDR librarieswas reconstructed to re-test the potential for affinity maturation inthe VH1 CDR. Residues 27, 28, and 30–32 were randomized using the newY0192 background.

Second-Generation Selections of Anti-VEGF Libraries: Based on Ala-scanresults as well as the crystal structure of the antigen-antibody(F(ab)-12) complex, a total of seventeen libraries were constructedusing the pY0192 template and stop-template oligonucleotides (which codefor a stop codon at the sites targeted for randomization) YC-80, YC-100,YC-102, HL-263, and HL-264 (Table 9 above). The correspondingrandomization oligonucleotides (which employ NNS at the sites targetedfor randomization) were YC81, YC-101, YC-103, HL-265, and HL-266 (Table10 above). The resulting transformants yielded libraries withcomplexities ranging from 6×10⁷ to 5×10⁸ which suggests that thelibraries were comprehensive in covering all possible variants. Phagelibraries were sorted for 7–8 rounds using conditions as described inTable 12 below.

TABLE 12 Conditions for Secondary Selections of Fab Variants Round ofIncubation Incubation Incubation Selection Time (hr) Solution Temp. (°C.) 1 0 0 room temp. 2 1 ELISA buffer room temp. 3 2 1 μM VEGF/ roomtemp. ELISA 4 18 1 μM VEGF/ room temp. ELISA 5 37 1 μM VEGF/ room temp.ELISA 6 17 hr @ room temp./ 1 μM VEGF/ room temp./ 30 hr @ 37° C. ELISA37° C. 7 63 1 μM VEGF/ 37° C. ELISA 8 121 1 μM VEGF/ 37° C. ELISA

ELISA buffer contained 0.5% bovine serum albumin and 0.05% TWEEN 20™ inPBS. VEGF was included in the incubation buffer to minimize rebinding ofphages to VEGF coated on the surface of the plate. Sorting of theselibraries yielded phage enrichments over 7 to 8 rounds of selection.

Phage-ELISA Assays of Second Generation Clones: After eight round ofselections, ten to twenty clones from each library were isolated fromcarbenicillin containing plates harboring E. coli (XL1) colonies whichhad been infected with an eluted phage pool. Colonies were isolated andgrown with helper phage to obtain single-stranded DNA for sequencing.CDR substitutions selected for more favorable binding to VEGF werededuced from the DNA sequences of phagemid clones. A sampling ofselected clones is shown in Table 13 below.

TABLE 13 Protein Sequences of Anti-VEGF Variants from Second GenerationFab-Phage Libraries Variants from library YC-81 Name VH3 sequence(residues 99–111) Y0238-1 YPYYRGTSHWYFD (SEQ ID NO:56) Y0238-2YPYYINKSHWYFD (SEQ ID NO:57) Y0238-3 YPYYYGTSHWYFD (SEQ ID NO:58)Y0238-4 YPYYYNQSHWYFD (SEQ ID NO:59) Y0238-5 YPYYIAKSHWYFD (SEQ IDNO:60) Y0238-6 YPYYRDNSHWYFD (SEQ ID NO:61) Y0238-7 YPYYWGTSHWYFD (SEQID NO:62) Y0238-8 YPYYRQNSHWYFD (SEQ ID NO:63) Y0238-9 YPYYRQSSHWYFD(SEQ ID NO:64) Y0238-10 YPYYRNTSHWYFD (SEQ ID NO:65) Y0238-11YPYYKNTSHWYFD (SEQ ID NO:66) Y0238-12 YPYYIERSHWYFD (SEQ ID NO:67)Y0228-21 YPYYRNASHWYFD (SEQ ID NO:68) Y0228-22 YPYYTTRSHWYFD (SEQ IDNO:69) Y0228-23 YPYYEGSSHWYFD (SEQ ID NO:70) Y0228-24 YPYYRQRGHWYFD (SEQID NO:71) Y0228-26 YPYYTGRSHWYFD (SEQ ID NO:72) Y0228-27 YPYYTNTSHWYFD(SEQ ID NO:73) Y0228-28 YPYYRKGSHWYFD (SEQ ID NO:74) Y0228-29YPYYTGSSHWYFD (SEQ ID NO:75) Y0228-30 YPYYRSGSHWYFD (SEQ ID NO:76)Y0229-20 YPYYTNRSHWYFD (SEQ ID NO:77) Y0229-21 YPYYRNSSHWYFD (SEQ IDNO:78) Y0229-22 YPYYKESSHWYFD (SEQ ID NO:79) Y0229-23 YPYYRDASHWYFD (SEQID NO:80) Y0229-24 YPYYRQKGHWYFD (SEQ ID NO:81) Y0229-25 YPYYKGGSHWYFD(SEQ ID NO:82) Y0229-26 YPYYYGASHWYFD (SEQ ID NO:83) Y0229-27YPYYRGESHWYFD (SEQ ID NO:84) Y0229-28 YPYYRSTSHWYFD (SEQ ID NO:85)Variants from library HL-265 Name VH1 sequence (residue 26–35) Y0243-1GYDFTHYGMN (5/10 clones) (SEQ ID NO:86) Y0243-2 GYEFQHYGMN (SEQ IDNO:87) Y0243-3 GYEFTHYGMN (SEQ ID NO:88) Y0243-4 GYDFGHYGMN (SEQ IDNO:89) Y0243-5 GYDFSHYGMN (SEQ ID NO:90) Y0243-6 GYEFSHYGMN (SEQ IDNO:91) Variants from library YC-101 Name VH “CDR7” sequence (residues70–79) Y0244-1 FSVDVSKSTA (SEQ ID NO:92) Y0244-2 FSLDKSKSTA (SEQ IDNO:93) Y0244-3 FSLDVWKSTA (SEQ ID NO:94) Y0244-4 FSIDKSKSTA (:95)The sequence of the randomized region only is shown as deduced from DNAsequencing.

When a number of clones were tested along with the parent clone pY0192in phage-ELISA assay, none showed a distinctive improvement over theparental clone. This could be explained by the time-scale on which theassay was performed (<3 hours).

In order to quantify improvement in antigen binding over parent clone,several anti-VEGF variants' DNA were transformed into E. coli strain34B8, expressed as Fab, and purified by passing the periplasmic shockatethrough a protein G column (Pharmacia) as described in Example 2 above.

CDR Combination Variants: To improve VEGF binding affinity further,mutations found by phage display were combined in different CDRs tocreate multiple-CDR mutants. In particular, the mutations identified inthe most affinity-improved phage variants from VH1, VH2, and VH3libraries were combined (Table 14) in order to test for additivity oftheir contributions to binding affinity.

TABLE 14 Combination CDR Anti-VEGF Variants Parent Mutagenesis oligo/Name clone comments Sequence Y0313-1 Y0243-1 YC-115 (VH3: H101Y5′-GCAAAGTACCCGTAGTATTATGGGAC (SEQ ID NO:96) and S105T)GAGCCACTGGTATTTC-3′ Y0317 Y0313-1 YC-108 (revert VL15′-GTCACCATCACCTGCAGCGCAAGTCA (SEQ ID NO:97) back to wild type)GGATATTAGCAACTATTTAAAC-3′ Y0313-3 Y0238-3 YC-116 (VH3;5′-CCGTACTATTATGGGAGCAGCCACTG (SEQ ID NO:98) T105S) GTATTTC-3′Mutations from the indicated parental vectors were combined with thosefrom the indicated oligonucleotide by site-directed mutagenesis to yieldthe combination variants listed.

Version Y0317 is equivalent to Y0313-1 except that the backgroundmutation in VL1 was removed and its sequence reverted back to that inpY0101. The effects of mutating H101Y and S105T were tested byconstructing a reversion mutant from Y0238-3.

BIAcore Analysis: The VEGF-binding affinities of Fab fragments werecalculated from association and dissociation rate constants measuredusing a BIAcore-2000™ surface plasmon resonance system (BIAcore, Inc.,Piscataway, N.J.). A biosensor chip was activated for covalent couplingof VEGF using N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's (BIAcore, Inc., Piscataway, N.J.) instructions. VEGF wasbuffered exchanged into 20 mM sodium acetate, pH 4.8 and diluted toapproximately 50 μg/mL. An aliquot (35 μL) was injected at a flow rateof 2 μL/min to achieve approximately 700–1400 response units (RU) ofcoupled protein. Finally, 1 M ethanolamine was injected as a blockingagent.

For kinetics measurements, two-fold serial dilutions of Fab wereinjected in PBS/TWEEN™ buffer (0.05% TWEEN 20™ in phosphate bufferedsaline) at 25° C. at a flow rate of 10 μL/min. On rates and off rateswere calculated using standard protocols (Karlsson et al. J. Immun.Methods 145:229–240 (1991)). Equilibrium dissociation constants, Kd'sfrom surface plasmon resonance (SPR) measurements were calculated askoff/kon. Data are shown in Table 15 below.

TABLE 15 Kinetics of Fab-VEGF binding from BIAcore ™ measurementsVariant Kon (10⁴/M/s) koff(10⁻⁴/s) Kd (nM) Kd (wt)/Kd Y0244-1 3.4 2.7 8   3.6 Y0244-4 5.2 1.7  3.3 0.9 Y0243-1 6.7 0.45 0.7 4.1 Y0238-3 1.7<0.04* <0.2* >14*   Y0238-7 1.5 <0.06* <0.4* >7.3* Y0238-10 1.6 0.09 0.64.8 Y0238-5 0.8 0.08 0.9 3.2 Y0238-1 2.6 0.09 0.4 7.3 Y0313-1 3.5  <0.054*   <0.15* >20*   Y0313-3 1.2  0.081  0.65 4.5 *The dissociationrate observed probably reflects an upper limit for the true dissociationrate in these experiments, since the off-rate is approaching the limitof detection by BIAcore.

The BIAcore™ data in Table 15 show that several variants had improvedaffinity over Y0192. For example, a CDRH1 variant, Y0243-1, showed 4.1fold enhanced affinity, arising from mutations T28D and N31H. VariantY0238-3 showed at least a 14 fold improvement in binding affinity overY0192. Both CDRH3 mutations contribute to the improved affinity ofY0238-3 because reversion of T105 to S (variant Y0313-3) reduces theaffinity of Y0238-3 from 0.15 nM to 0.65 nM (see Table 15). The greateraffinity enhancement relative to Y0192 was seen for Y0313-1, whichcontained CDRH3 mutations combined with CDRH1 mutations.

Cell-Based Assay of VEGF Inhibition: Several versions of the A4.6.1anti-VEGF antibody were tested for their ability to antagonize VEGF(recombinant; version 1-165) in induction of the growth of HuVECs (humanumbilical vein endothelial cells). The 96-well plates were seeded with1000 HuVECs per well and fasted in assay medium (F12:DMEM 50:50supplemented with 1.5% diafiltered fetal bovine serum) for 24 h. Theconcentration of VEGF used for inducing the cells was determined byfirst titrating for the amount of VEGF that can stimulate 80% of maximalDNA synthesis. Fresh assay medium containing fixed amounts of VEGF (0.2nM final concentration), and increasing concentrations of anti-VEGF Fabor Mab were then added. After 40 h of incubation, DNA synthesis wasmeasured by incorporation of tritiated thymidine. Cells were pulsed with0.5 μCi per well of [3H]-thymidine for 24 h and harvested for counting,using a TopCount gamma counter.

The results (FIG. 11) show that the full-length IgG form of F(ab)-12 wassignificantly more potent in inhibiting VEGF activity than the Fab form(here, Y0192 was used). However, both variants Y0238-3 and Y0313-1showed even more potent inhibition of VEGF activity than either theY0192 Fab or F(ab)-12 Mab. Comparing the Fab forms, variant Y0313-1appeared >30-fold more potent than the wild-type Fab. It should be notedthat the amount of VEGF (0.2 nM) used in this assay is potentiallylimiting for determination of an accurate IC50 for the mutant. Forexample, if the binding affinity (Kd) of the mutant is in fact <0.2 nM,the IC50 in this experiment will appear higher than under conditions oflower VEGF concentration. The result therefore supports the conclusionthat the affinity-improved variant is at least 30-fold improved inaffinity for VEGF, and that it effectively blocks VEGF activity invitro. Since the variant Y0317 differs from Y0313-1 only in thereversion of the VL1 sequence to wild-type (FIG. 10A), it is predictedthat Y0317 will have similar activity to Y0313-1.

Variant Y0317 (Fab) and humanized variant F(ab)-12 from Example 1 (fulllength and Fab) were compared for their ability to inhibit bovinecapillary endothelial cell proliferation in response to a near maximallyeffective concentration of VEGF using the assay described in Example 1.As illustrated in FIG. 12, Y0317 was markedly more effective atinhibiting bovine capillary endothelial cell proliferation than the fulllength and Fab forms of F(ab)-12 in this assay. The Y0317 affinitymatured Fab demonstrated an ED50 value in this assay which was at leastabout 20 fold lower than F(ab)-12 Fab.

1. A humanized anti-vascular endothelial growth factor (VEGF) antibodyor an antigen binding fragment thereof which binds human VEGF with a Kdvalue of no more than about 1×10⁻⁸M, said humanized anti-VEGF antibodyhaving a heavy a chain variable domain comprising the following heavychain complementarity determining region (CDR) amino acid sequences:CDRH1 (GYX1FTX2YGMN), wherein X1 is T or D and X2 is N or H; SEQ IDNO:130), CDRH2 (WINTYTGEPTYAADFKR; SEQ ID NO:2) and CDRH3(YPX1YYGX2SHWYFDV, wherein X1 is Y or H and X2 is S or T; SEQ IDNO:131).
 2. The humanized anti-VEGF antibody of claim 1, wherein theheavy chain variable domain comprises the amino acid sequence of SEQ IDNO:7.
 3. The humanized anti-VEGF antibody of claim 1 having a heavychain variable domain comprising the following heavy chain CDR aminoacid sequences: CDRH1 (GYTFTNYGMN; SEQ ID NO:1), CDRH2(WINTYTGEPTYAADFKR; SEQ ID NO:2) and CDRH3 (YPHYYGSSHWYFDV; SEQ IDNO:3).
 4. The humanized anti-VEGF antibody of claim 1 having a lightchain variable domain comprising the following light chain CDR aminoacid sequences: CDRL1 (SASQDISNYLN; SEQ ID NO:4), CDRL2 (FTSSLHS; SEQ IDNO:5) and CDRL3 (QQYSTVPWT; SEQ ID NO:6).
 5. The humanized anti-VEGFantibody of claim 4, wherein the light chain variable domain comprisesthe amino acid sequence of SEQ ID NO:8.
 6. The humanized anti-VEGFantibody of claim 1 having a heavy chain variable domain comprising theamino acid sequence of SEQ ID NO:7 and a light chain variable domaincomprising the amino acid sequence of SEQ ID NO:8.
 7. The humanizedanti-VEGF antibody of claim 1 which is a full length antibody.
 8. Thehumanized anti-VEGF antibody of claim 7 which is a human IgG.
 9. Thehumanized anti-VEGF antibody of claim 1 which is an antigen bindingfragment.
 10. The antibody fragment of claim 9 which is a Fab.
 11. Acomposition comprising the humanized anti-VEGF antibody of claim 1 and apharmaceutically acceptable carrier.
 12. The humanized anti-VEGFantibody of claim 1 having a heavy chain variable domain comprising theamino acid sequence of SEQ ID NO:118 and a light chain variable domaincomprising the amino acid sequence of SEQ ID NO:117.